Microorganisms for the production of insect pheromones and related compounds

ABSTRACT

The present application relates to recombinant microorganisms useful in the biosynthesis of unsaturated C6-C24 fatty alcohols, aldehydes, and acetates which may be useful as insect pheromones, fragrances, flavors, and polymer intermediates. The C6-C24 fatty alcohols, aldehydes, and acetates described herein may be used as substrates for metathesis reactions to expand the repertoire of target compounds and pheromones. The application further relates to recombinant microorganisms co-expressing a pheromone pathway and a pathway for the production of a toxic protein, peptide, oligonucleotide, or small molecule suitable for use in an attract-and-kill pest control approach. Also provided are methods of producing unsaturated C6-C24 fatty alcohols, aldehydes, and acetates using the recombinant microorganisms, as well as compositions comprising the recombinant microorganisms and/or optionally one or more of the product alcohols, aldehydes, or acetates.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of U.S. patent application Ser. No.16/420,566, filed May 23, 2019 (issued as U.S. Pat. No. 11,109,596 onSep. 7, 2021), which is a Continuation of U.S. patent application Ser.No. 15/983,706 filed May 18, 2018 (issued as U.S. Pat. No. 10,308,962 onJun. 4, 2019), which is a Continuation of International Application No.PCT/US2016/062852, filed on Nov. 18, 2016, which claims the benefit ofpriority to U.S. Provisional Application No. 62/257,054, filed Nov. 18,2015, and U.S. Provisional Application No. 62/351,605, filed Jun. 17,2016; each of the aforementioned applications is herein expresslyincorporated by reference.

STATEMENT REGARDING THE SEQUENCE LISTING

The Sequence Listing associated with this application is provided intext format in lieu of a paper copy, and is hereby incorporated byreference into the specification. The name of the text file containingthe Sequence Listing is PRVI_007_05US_SeqList_ST25.txt. The text file isabout 56 KB, was created on PRVI_007_05US_SeqList_ST25.txt, and is beingsubmitted electronically via EFS-Web.

TECHNICAL FIELD

This application relates to recombinant microorganisms useful in thebiosynthesis of unsaturated C₆-C₂₄ fatty alcohols, aldehydes, andacetates which may be useful as insect pheromones, fragrances, flavors,and polymer intermediates. The application further relates to methods ofproducing unsaturated C₆-C₂₄ fatty alcohols, aldehydes, and acetatesusing the recombinant microorganisms, as well as compositions comprisingone or more of these compounds and/or the recombinant microorganisms.

BACKGROUND

As the global demand for food grows, there is an increasing need foreffective pest control. Conventional insecticides are among the mostpopular chemical control agents because they are readily available,rapid acting, and highly reliable. However, the overuse, misuse, andabuse of these chemicals have led to resistant pests, alteration of thenatural ecology, and in some cases, environmental damage.

The use of insect pheromones to control pest populations has gainedincreasing popularity as a viable, safe, and environmentally-friendlyalternative to conventional insecticides. Since their discovery in thelate 1950s, these molecules have shown efficacy in reducing insectpopulations through a variety of methods, including mass trappings,attract and kill, and mating disruption. The latter method in particularrepresents a non-toxic means of pest control and utilizes the ability ofsynthetic pheromones to mask naturally occurring pheromones, therebycausing confusion and mating disruption.

Although pheromones have significant potential in agricultural insectcontrol, the cost of synthesizing pheromones using currently availabletechniques is very high, which prohibits widespread use of thissustainable technology beyond high-value crops. Thus, there is anexisting need to develop novel technologies for the cost-efficientproduction of insect pheromones and related fragrances, flavors, andpolymer intermediates. The present inventors address this need with thedevelopment of recombinant microorganisms capable of producing awide-range of unsaturated C₆-C₂₄ fatty alcohols, aldehydes, and acetatesincluding synthetic insect pheromones from low-cost feedstocks.

SUMMARY OF THE DISCLOSURE

The present application relates to recombinant microorganisms having abiosynthesis pathway for the production of one or more compoundsselected from unsaturated C₆-C₂₄ fatty alcohols, aldehydes, andacetates. The recombinant microorganisms described herein may be usedfor the production of at least one compound, such as an insectpheromone, a fragrance, or a flavoring agent, selected from unsaturatedC₆-C₂₄ fatty alcohols, aldehydes, and acetates.

In one embodiment, the recombinant microorganism comprises abiosynthesis pathway for the production of an unsaturated C₆-C₂₄ fattyalcohol. Accordingly, in a first aspect, the application relates to arecombinant microorganism capable of producing an unsaturated C₆-C₂₄fatty alcohol from an endogenous or exogenous source of saturated C₆-C₂₄fatty acyl-CoA, wherein the recombinant microorganism expresses (a): atleast one exogenous nucleic acid molecule encoding a fatty-acyldesaturase that catalyzes the conversion of a saturated C₆-C₂₄ fattyacyl-CoA to a corresponding mono- or poly-unsaturated C₆-C₂₄ fattyacyl-CoA; and (b): at least one exogenous nucleic acid molecule encodinga fatty alcohol forming fatty-acyl reductase that catalyzes theconversion of the mono- or poly-unsaturated C₆-C₂₄ fatty acyl-CoA from(a) into the corresponding mono- or poly-unsaturated C₆-C₂₄ fattyalcohol. In some embodiments, the mono- or poly-unsaturated C₆-C₂₄ fattyalcohol is an insect pheromone. In some embodiments, the mono- orpoly-unsaturated C₆-C₂₄ fatty alcohol is a fragrance or flavoring agent.In some embodiments, the recombinant microorganism further comprises atleast one endogenous or exogenous nucleic acid molecule encoding analcohol oxidase or an alcohol dehydrogenase, wherein the alcohol oxidaseor alcohol dehydrogenase is capable of catalyzing the conversion of themono- or poly-unsaturated C₆-C₂₄ fatty alcohol from (b) into acorresponding mono- or poly-unsaturated C₆-C₂₄ fatty aldehyde. In someembodiments, the recombinant microorganism further comprises at leastone endogenous or exogenous nucleic acid molecule encoding an acetyltransferase capable of catalyzing the conversion of the mono- orpoly-unsaturated C₆-C₂₄ fatty alcohol from (b) into a correspondingmono- or poly-unsaturated C₆-C₂₄ fatty acetate.

In some embodiments, the fatty-acyl desaturase is a desaturase capableof utilizing a fatty acyl-CoA as a substrate that has a chain length of6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or24 carbon atoms.

In some embodiments, the fatty-acyl desaturase is capable of generatinga double bond at position C5, C6, C7, C8, C9, C10, C11, C12, or C13 inthe fatty acid or its derivatives, such as, for example, fatty acid CoAesters.

In one exemplary embodiment, the fatty-acyl desaturase is a Z11desaturase. In various embodiments described herein, the Z11 desaturase,or the nucleic acid sequence that encodes it, can be isolated fromorganisms of the species Agrotis segetum, Amyelois transitella,Argyrotaenia velutiana, Choristoneura rosaceana, Lampronia capitella,Trichoplusia ni, Helicoverpa zea, or Thalassiosira pseudonana. FurtherZ11-desaturases, or the nucleic acid sequences encoding them, can beisolated from Bombyx mori, Manduca sexta, Dicaraeo grandiosella, Eariasinsulana, Earias vittella, Plutella xylostella, Bombyx mori or Diaphanianitidalis. In exemplary embodiments, the Z11 desaturase comprises asequence selected from GenBank Accession Nos. JX679209, JX964774,AF416738, AF545481, EU152335, AAD03775, AAF81787 (SEQ ID NO: 39), andAY493438. In some embodiments, a nucleic acid sequence encoding a Z11desaturase from organisms of the species Agrotis segetum, Amyeloistransitella, Argyrotaenia velutiana, Choristoneura rosaceana, Lamproniacapitella, Trichoplusia ni, Helicoverpa zea, or Thalassiosira pseudonanais codon optimized. In some embodiments, the Z11 desaturase comprises asequence selected from SEQ ID NOs: 9, 18, 24 and 26 from Trichoplusiani. In other embodiments, the Z11 desaturase comprises a sequenceselected from SEQ ID NOs: 10 and 16 from Agrotis segetum. In someembodiments, the Z11 desaturase comprises a sequence selected from SEQID NOs: 11 and 23 from Thalassiosira pseudonana. In certain embodiments,the Z11 desaturase comprises a sequence selected from SEQ ID NOs: 12, 17and 30 from Amyelois transitella. In further embodiments, the Z11desaturase comprises a sequence selected from SEQ ID NOs: 13, 19, 25, 27and 31 from Helicoverpa zea. In some embodiments, the Z11 desaturasecomprises a chimeric polypeptide. In some embodiments, a complete orpartial Z11 desaturase is fused to another polypeptide. In certainembodiments, the N-terminal native leader sequence of a Z11 desaturaseis replaced by an oleosin leader sequence from another species. Incertain embodiments, the Z11 desaturase comprises a sequence selectedfrom SEQ ID NOs: 15, 28 and 29.

In certain embodiments, the Z11 desaturase catalyzes the conversion of afatty acyl-CoA into a mono- or poly-unsaturated product selected fromZ11-13:Acyl-CoA, E11-13:Acyl-CoA, (Z,Z)-7,11-13:Acyl-CoA,Z11-14:Acyl-CoA, E11-14:Acyl-CoA, (E,E)-9,11-14:Acyl-CoA,(E,Z)-9,11-14:Acyl-CoA, (Z,E)-9,11-14:Acyl-CoA, (Z,Z)-9,11-14:Acyl-CoA,(E,Z)-9,11-15:Acyl-CoA, (Z,Z)-9,11-15:Acyl-CoA, Z11-16:Acyl-CoA,E11-16:Acyl-CoA, (E,Z)-6,11-16:Acyl-CoA, (E,Z)-7,11-16:Acyl-CoA,(E,Z)-8,11-16:Acyl-CoA, (E,E)-9,11-16:Acyl-CoA, (E,Z)-9,11-16:Acyl-CoA,(Z,E)-9,11-16:Acyl-CoA, (Z,Z)-9,11-16:Acyl-CoA, (E,E)-11,13-16:Acyl-CoA,(E,Z)-11,13-16:Acyl-CoA, (Z,E)-11,13-16:Acyl-CoA,(Z,Z)-11,13-16:Acyl-CoA, (Z,E)-11,14-16:Acyl-CoA,(E,E,Z)-4,6,11-16:Acyl-CoA, (Z,Z,E)-7,11,13-16:Acyl-CoA,(E,E,Z,Z)-4,6,11,13-16:Acyl-CoA, Z11-17:Acyl-CoA,(Z,Z)-8,11-17:Acyl-CoA, Z11-18:Acyl-CoA, E11-18:Acyl-CoA,(Z,Z)-11,13-18:Acyl-CoA, (E,E)-11,14-18:Acyl-CoA, or combinationsthereof.

In another exemplary embodiment, the fatty-acyl desaturase is a Z9desaturase. In various embodiments described herein, the Z9 desaturase,or the nucleic acid sequence that encodes it, can be isolated fromorganisms of the species Ostrinia furnacalis, Ostrinia nobilalis,Choristoneura rosaceana, Lampronia capitella, Helicoverpa assulta, orHelicoverpa zea. In exemplary embodiments, the Z9 desaturase comprises asequence selected from GenBank Accession Nos. AY057862, AF243047,AF518017, EU152332, AF482906, and AAF81788. In some embodiments, anucleic acid sequence encoding a Z9 desaturase is codon optimized. Insome embodiments, the Z9 desaturase comprises a sequence set forth inSEQ ID NO: 20 from Ostrinia furnacalis. In other embodiments, the Z9desaturase comprises a sequence set forth in SEQ ID NO: 21 fromLampronia capitella. In some embodiments, the Z9 desaturase comprises asequence set forth in SEQ ID NO: 22 from Helicoverpa zea.

In certain embodiments, the Z9 desaturase catalyzes the conversion of afatty acyl-CoA into a monounsaturated or polyunsaturated productselected from Z9-11:Acyl-CoA, Z9-12:Acyl-CoA, E9-12:Acyl-CoA,(E,E)-7,9-12:Acyl-CoA, (E,Z)-7,9-12:Acyl-CoA, (Z,E)-7,9-12:Acyl-CoA,(Z,Z)-7,9-12:Acyl-CoA, Z9-13:Acyl-CoA, E9-13:Acyl-CoA,(E,Z)-5,9-13:Acyl-CoA, (Z,E)-5,9-13:Acyl-CoA, (Z,Z)-5,9-13:Acyl-CoA,Z9-14:Acyl-CoA, E9-14:Acyl-CoA, (E,Z)-4,9-14:Acyl-CoA,(E,E)-9,11-14:Acyl-CoA, (E,Z)-9,11-14:Acyl-CoA, (Z,E)-9,11-14:Acyl-CoA,(Z,Z)-9,11-14:Acyl-CoA, (E,E)-9,12-14:Acyl-CoA, (Z,E)-9,12-14:Acyl-CoA,(Z,Z)-9,12-14:Acyl-CoA, Z9-15:Acyl-CoA, E9-15:Acyl-CoA,(Z,Z)-6,9-15:Acyl-CoA, Z9-16:Acyl-CoA, E9-16:Acyl-CoA,(E,E)-9,11-16:Acyl-CoA, (E,Z)-9,11-16:Acyl-CoA, (Z,E)-9,11-16:Acyl-CoA,(Z,Z)-9,11-16:Acyl-CoA, Z9-17:Acyl-CoA, E9-18:Acyl-CoA, Z9-18:Acyl-CoA,(E,E)-5,9-18:Acyl-CoA, (E,E)-9,12-18:Acyl-CoA, (Z,Z)-9,12-18:Acyl-CoA,(Z,Z,Z)-3,6,9-18:Acyl-CoA, (E,E,E)-9,12,15-18:Acyl-CoA,(Z,Z,Z)-9,12,15-18:Acyl-CoA, or combinations thereof.

In some embodiments, the recombinant microorganism may express abifunctional desaturase capable of catalyzing the subsequentdesaturation of two double bonds.

In some embodiments, the recombinant microorganism may express more thanone exogenous nucleic acid molecule encoding a fatty-acyl desaturasethat catalyzes the conversion of a saturated C₆-C₂₄ fatty acyl-CoA to acorresponding mono- or poly-unsaturated C₆-C₂₄ fatty acyl-CoA. Forinstance, the recombinant microorganism may express an exogenous nucleicacid molecule encoding a Z11 desaturase and another exogenous nucleicacid molecule encoding a Z9 desaturase.

In some embodiments, the recombinant microorganism may express afatty-acyl conjugase that acts independently or together with afatty-acyl desaturase to catalyze the conversion of a saturated ormonounsaturated fatty acyl-CoA to a conjugated polyunsaturated fattyacyl-CoA.

In one embodiment, the disclosure provides a recombinant microorganismcapable of producing a polyunsaturated C₆-C₂₄ fatty alcohol from anendogenous or exogenous source of saturated or monounsaturated C₆-C₂₄fatty acyl-CoA, wherein the recombinant microorganism expresses: (a) atleast one exogenous nucleic acid molecule encoding a fatty acylconjugase that catalyzes the conversion of a saturated ormonounsaturated C₆-C₂₄ fatty acyl-CoA to a corresponding polyunsaturatedC₆-C₂₄ fatty acyl-CoA; and (b) at least one exogenous nucleic acidmolecule encoding a fatty alcohol forming fatty-acyl reductase thatcatalyzes the conversion of the polyunsaturated C₆-C₂₄ fatty acyl-CoAfrom (a) into the corresponding polyunsaturated C₆-C₂₄ fatty alcohol.

In another embodiment, the recombinant microorganism expresses at leasttwo exogenous nucleic acid molecules encoding fatty-acyl conjugases thatcatalyze the conversion of a saturated or monounsaturated C₆-C₂₄ fattyacyl-CoA to a corresponding polyunsaturated C₆-C₂₄ fatty acyl-CoA.

In a further embodiment, the disclosure provides a recombinantmicroorganism capable of producing a polyunsaturated C₆-C₂₄ fattyalcohol from an endogenous or exogenous source of saturated ormonounsaturated C₆-C₂₄ fatty acyl-CoA, wherein the recombinantmicroorganism expresses: (a) at least one exogenous nucleic acidmolecule encoding a fatty-acyl desaturase and at least one exogenousnucleic acid molecule encoding a fatty acyl conjugase that catalyze theconversion of a saturated or monounsaturated C₆-C₂₄ fatty acyl-CoA to acorresponding polyunsaturated C₆-C₂₄ fatty acyl-CoA; and (b) at leastone exogenous nucleic acid molecule encoding a fatty alcohol formingfatty-acyl reductase that catalyzes the conversion of thepolyunsaturated C₆-C₂₄ fatty acyl-CoA from (a) into the correspondingpolyunsaturated C₆-C₂₄ fatty alcohol.

In another embodiment, the recombinant microorganism expresses at leasttwo exogenous nucleic acid molecules encoding fatty-acyl desaturases andat least two exogenous nucleic acid molecules encoding fatty-acylconjugases that catalyze the conversion of a saturated ormonounsaturated C₆-C₂₄ fatty acyl-CoA to a corresponding polyunsaturatedC₆-C₂₄ fatty acyl-CoA.

In yet a further embodiment, the fatty-acyl conjugase is a conjugasecapable of utilizing a fatty acyl-CoA as a substrate that has a chainlength of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, or 24 carbon atoms.

In certain embodiments, the conjugase, or the nucleic acid sequence thatencodes it, can be isolated from organisms of the species Cydiapomonella, Cydia nigricana, Lobesia botrana, Myelois cribrella, Plodiainterpunctella, Dendrolimus punctatus, Lampronia capitella, Spodopteralitura, Amyelois transitella, Manduca sexta, Bombyx mori, Calendulaofficinalis, Trichosanthes kirilowii, Punica granatum, Momordicacharantia, Impatiens balsamina, and Epiphyas postvittana. In exemplaryembodiments, the conjugase comprises a sequence selected from GenBankAccession No. or Uniprot database: A0A059TBF5, A0A0M3L9E8, A0A0M3L9S4,A0A0M3LAH8, A0A0M3LAS8, A0A0M3LAH8, B6CBS4, XP_013183656.1,XP_004923568.2, ALA65425.1, NP_001296494.1, NP_001274330.1, Q4A181,Q75PL7, Q9FPP8, AY178444, AY178446, AF182521, AF182520, Q95UJ3.

In various embodiments described herein, the fatty alcohol formingacyl-CoA reductase, i.e., fatty alcohol forming fatty-acyl reductase, orthe nucleic acid sequence that encodes it, can be isolated fromorganisms of the species Agrotis segetum, Spodoptera littoralis, orHelicoverpa amigera. In exemplary embodiments, the reductase comprises asequence selected from GenBank Accession Nos. JX679210 and HG423128, andUniProt Accession No. I3PN86. In some embodiments, a nucleic acidsequence encoding a fatty-acyl reductase from organisms of the speciesAgrotis segetum, Spodoptera littoralis, or Helicoverpa amigera is codonoptimized. In some embodiments, the reductase comprises a sequence setforth in SEQ ID NO: 1 from Agrotis segetum. In other embodiments, thereductase comprises a sequence set forth in SEQ ID NO: 2 from Spodopteralittoralis. In some embodiments, the reductase comprises a sequenceselected from SEQ ID NOs: 3 and 32 from Helicoverpa armigera.

In certain embodiments, the fatty alcohol forming fatty-acyl reductasecatalyzes the conversion of a mono- or poly-unsaturated fatty acyl-CoAinto a fatty alcohol product selected from (Z)-3-hexenol, (Z)-3-nonenol,(Z)-5-decenol, (E)-5-decenol, (Z)-7-dodecenol, (E)-8-dodecenol,(Z)-8-dodecenol, (Z)-9-dodecenol, (Z)-9-tetradecenol, (Z)-9-hexadecenol,(Z)-11-tetradecenol, (Z)-7-hexadecenol, (Z)-11-hexadecenol,(E)-11-tetradecenol, or (Z,Z)-11,13-hexadecadienol,(11Z,13E)-hexadecadienol, (E,E)-8,10-dodecadienol,(E,Z)-7,9-dodecadienol, (Z)-13-octadecenol, or combinations thereof.

In some embodiments, the recombinant microorganism may express more thanone exogenous nucleic acid molecule encoding a fatty alcohol formingfatty-acyl reductase that catalyzes the conversion of a mono- orpoly-unsaturated C₆-C₂₄ fatty acyl-CoA to a corresponding mono- orpoly-unsaturated C₆-C₂₄ fatty alcohol. Such recombinant microorganismsmay be advantageously used to produce blends of various insectpheromones.

In addition to the biosynthetic pathway described in the first aspectabove, the present application provides an additional biosyntheticpathway for the production of an unsaturated C₆-C₂₄ fatty alcoholutilizing a saturated C₆-C₂₄ fatty acyl-ACP intermediate derived from aC₆-C₂₄ fatty acid. Accordingly, in a second aspect, the applicationrelates to a recombinant microorganism capable of producing anunsaturated C₆-C₂₄ fatty alcohol from an endogenous or exogenous sourceof C₆-C₂₄ fatty acid, wherein the recombinant microorganism expresses(a): at least one exogenous nucleic acid molecule encoding an acyl-ACPsynthetase that catalyzes the conversion of a C₆-C₂₄ fatty acid to acorresponding saturated C₆-C₂₄ fatty acyl-ACP; (b) at least oneexogenous nucleic acid molecule encoding a fatty-acyl-ACP desaturasethat catalyzes the conversion of a saturated C₆-C₂₄ fatty acyl-ACP to acorresponding mono- or poly-unsaturated C₆-C₂₄ fatty acyl-ACP; (c) oneor more endogenous or exogenous nucleic acid molecules encoding a fattyacid synthase complex that catalyzes the conversion of the mono- orpoly-unsaturated C₆-C₂₄ fatty acyl-ACP from (b) to a corresponding mono-or poly-unsaturated C₆-C₂₄ fatty acyl-ACP with a two carbon elongationrelative to the product of (b); (d): at least one exogenous nucleic acidmolecule encoding a fatty aldehyde forming fatty-acyl reductase thatcatalyzes the conversion of the mono- or poly-unsaturated C₆-C₂₄ fattyacyl-ACP from (c) into a corresponding mono- or poly-unsaturated C₆-C₂₄fatty aldehyde; and (e) at least one endogenous or exogenous nucleicacid molecule encoding a dehydrogenase that catalyzes the conversion ofthe mono- or poly-unsaturated C₆-C₂₄ fatty aldehyde C₆-C₂₄ from (d) intoa corresponding mono- or poly-unsaturated C₆-C₂₄ fatty alcohol. In someembodiments, the mono- or poly-unsaturated C₆-C₂₄ fatty alcohol is aninsect pheromone. In some embodiments, the mono- or poly-unsaturatedC₆-C₂₄ fatty alcohol is a fragrance or flavoring agent. In someembodiments, the recombinant microorganism further comprises at leastone endogenous or exogenous nucleic acid molecule encoding an alcoholoxidase or an alcohol dehydrogenase, wherein the alcohol oxidase oralcohol dehydrogenase is capable of catalyzing the conversion of themono- or poly-unsaturated C₆-C₂₄ fatty alcohol from (e) into acorresponding mono- or poly-unsaturated C₆-C₂₄ fatty aldehyde. In someembodiments, the recombinant microorganism further comprises at leastone endogenous or exogenous nucleic acid molecule encoding an acetyltransferase capable of catalyzing the conversion of the mono- orpoly-unsaturated C₆-C₂₄ fatty alcohol from (e) into a correspondingmono- or poly-unsaturated C₆-C₂₄ fatty acetate.

In some embodiments, acyl-ACP synthetase is a synthetase capable ofutilizing a fatty acid as a substrate that has a chain length of 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24carbon atoms.

In various embodiments described herein, the acyl-ACP synthetase, or thenucleic acid that encodes it, can be isolated from organisms of thespecies Vibrio harveyi, Rhodotorula glutinis, or Yarrowia lipolytica.

In some embodiments, the fatty-acyl-ACP desaturase is a solubledesaturase. In various embodiments described herein, the fatty-acyl-ACPdesaturase, or the nucleic acid that encodes it, can be isolated fromorganisms of the species Pelargonium hortorum, Asclepias syriaca, orUncaria tomentosa.

In some embodiments, the recombinant microorganism may express more thanone exogenous nucleic acid molecule encoding a fatty-acyl desaturasethat catalyzes the conversion of a saturated C₆-C₂₄ fatty acyl-ACP to acorresponding mono- or poly-unsaturated C₆-C₂₄ fatty acyl-ACP.

As described above, fatty acid elongation enzymes, i.e., a fatty acidsynthase complex, can be utilized to extend the chain length of a mono-or poly-unsaturated C₆-C₂₄ fatty acyl-ACP by two additional carbons atthe alpha carbon. In some embodiments, the two additional carbons arederived from endogenous malonyl-CoA. In one embodiment, the one or morenucleic acid molecules encoding a fatty acid synthase complex areendogenous nucleic acid molecules, i.e., the nucleic acid molecule(s)is/are native to the recombinant microorganism. In another embodiment,the one or more nucleic acid molecules encoding a fatty acid synthasecomplex are exogenous nucleic acid molecules.

In various embodiments described herein, the fatty aldehyde formingacyl-ACP reductase, i.e., fatty aldehyde forming fatty-acyl reductase,or the nucleic acid sequence that encodes it, can be isolated fromorganisms of the species can be isolated from organisms of the speciesPelargonium hortorum, Asclepias syriaca, and Uncaria tomentosa.

In some embodiments, the recombinant microorganism may express more thanone exogenous nucleic acid molecule encoding a fatty aldehyde formingfatty-acyl reductase that catalyzes the conversion of a mono- orpoly-unsaturated C₆-C₂₄ fatty acyl-ACP to a corresponding mono- orpoly-unsaturated C₆-C₂₄ fatty aldehyde. Such recombinant microorganismsmay be advantageously used to produce blends of various insectpheromones. An exemplary blend according to the instant inventioncomprises of (Z)-11-hexadecenal (Z11-16:Ald) and (Z)-9-hexadecenal(Z9-16:Ald). In one embodiment, the ratio of the blend is 90:10, 91:9,92:8, 93:7, 94:6, 95:5, 96:4, 97:3, 98:2, or 99:1 ratio of(Z)-11-hexadecenal (Z11-16:Ald) to (Z)-9-hexadecenal (Z9-16:Ald). In anexemplary embodiment, the blend is a 97:3 ratio of (Z)-11-hexadecenal(Z11-16:Ald) to (Z)-9-hexadecenal (Z9-16:Ald), corresponding to keycomponents of the Helicoverpa female virgin.

As noted above, the recombinant microorganism according to the secondaspect comprises at least one endogenous or exogenous nucleic acidmolecule encoding a dehydrogenase capable of catalyzing the conversionof the mono- or poly-unsaturated C₆-C₂₄ fatty aldehyde from (d) into acorresponding mono- or poly-unsaturated C₆-C₂₄ fatty alcohol. In oneembodiment, the dehydrogenase is encoded by an endogenous nucleic acidmolecule. In another embodiment, the dehydrogenase is encoded by anexogenous nucleic acid molecule. In exemplary embodiments, theendogenous or exogenous nucleic acid molecule encoding a dehydrogenaseis isolated from organisms of the species Saccharomyces cerevisiae,Escherichia coli, Yarrowia lipolytica, or Candida tropicalis.

In addition to the biosynthetic pathway described in the first andsecond aspects above, the present application provides an additionalbiosynthetic pathway for the production of an unsaturated C₆-C₂₄ fattyalcohol utilizing a saturated C₆-C₂₄ fatty acyl-ACP intermediate derivedfrom a C₆-C₂₄ fatty acid. Accordingly, in a third aspect, theapplication relates to a recombinant microorganism capable of producingan unsaturated C₆-C₂₄ fatty alcohol from an endogenous or exogenoussource of C₆-C₂₄ fatty acid, wherein the recombinant microorganismexpresses (a): at least one exogenous nucleic acid molecule encoding anacyl-ACP synthetase that catalyzes the conversion of a C₆-C₂₄ fatty acidto a corresponding saturated C₆-C₂₄ fatty acyl-ACP; (b) at least oneexogenous nucleic acid molecule encoding a fatty-acyl-ACP desaturasethat catalyzes the conversion of a saturated C₆-C₂₄ fatty acyl-ACP to acorresponding mono- or poly-unsaturated C₆-C₂₄ fatty acyl-ACP; (c) atleast one exogenous fatty acyl-ACP thioesterase that catalyzes theconversion of the mono- or poly-unsaturated C₆-C₂₄ fatty acyl-ACP from(b) to a corresponding mono- or poly-unsaturated C₆-C₂₄ fatty acid; (d)one or more endogenous or exogenous nucleic acid molecules encoding anelongase that catalyzes the conversion of the mono- or poly-unsaturatedC₆-C₂₄ fatty acyl-CoA derived from CoA activation of the mono- orpoly-unsaturated C₆-C₂₄ fatty acid from (c) to a corresponding mono- orpoly-unsaturated C₆-C₂₄ fatty acyl-CoA with a two carbon or greaterelongation relative to the product of (c); and (e): at least oneexogenous nucleic acid molecule encoding a fatty alcohol formingfatty-acyl reductase that catalyzes the conversion of the mono- orpoly-unsaturated C₆-C₂₄ fatty acyl-CoA from (d) into a correspondingmono- or poly-unsaturated C₆-C₂₄ fatty alcohol. In some embodiments, themono- or poly-unsaturated C₆-C₂₄ fatty alcohol is an insect pheromone.In some embodiments, the mono- or poly-unsaturated C₆-C₂₄ fatty alcoholis a fragrance or flavoring agent. In some embodiments, the recombinantmicroorganism further comprises at least one endogenous or exogenousnucleic acid molecule encoding an alcohol oxidase or an alcoholdehydrogenase, wherein the alcohol oxidase or alcohol dehydrogenase iscapable of catalyzing the conversion of the mono- or poly-unsaturatedC₆-C₂₄ fatty alcohol from (e) into a corresponding mono- orpoly-unsaturated C₆-C₂₄ fatty aldehyde. In some embodiments, therecombinant microorganism further comprises at least one endogenous orexogenous nucleic acid molecule encoding an acetyl transferase capableof catalyzing the conversion of the mono- or poly-unsaturated C₆-C₂₄fatty alcohol from (e) into a corresponding mono- or poly-unsaturatedC₆-C₂₄ fatty acetate

In some embodiments according to this third aspect, a fatty acyl-ACPthioesterase can be utilized to convert a mono- or poly-unsaturatedC₆-C₂₄ fatty acyl-ACP into a corresponding mono- or poly-unsaturatedC₆-C₂₄ fatty acid. In a particular embodiment, soluble fatty acyl-ACPthioesterases can be used to release free fatty acids for reactivationto a CoA thioester. Fatty acyl-ACP thioesterases including Q41635,Q39473, P05521.2, AEM72519, AEM72520, AEM72521, AEM72523, AAC49784,CAB60830, EER87824, EER96252, ABN54268, AA077182, CAH09236, ACL08376,and homologs thereof may be used. In some embodiments, the mono- orpoly-unsaturated C₆-C₂₄ fatty acyl-CoA may serve as a substrate for anelongase, which can be utilized to extend the chain length of a mono- orpoly-unsaturated C₆-C₂₄ fatty acyl-CoA by two additional carbons at thealpha carbon. In some embodiments, the two additional carbons arederived from endogenous malonyl-CoA.

As described above, in some embodiments, the recombinant microorganismaccording to the first, second, or third aspect further comprises atleast one endogenous or exogenous nucleic acid molecule encoding analcohol oxidase capable of catalyzing the conversion of a mono- orpoly-unsaturated C₆-C₂₄ fatty alcohol into a corresponding mono- orpoly-unsaturated C₆-C₂₄ fatty aldehyde. In certain embodiments, thealcohol oxidase, or the nucleic acid sequence that encodes it, can beisolated from organisms of the species Candida boidinii, Komagataellapastoris, Tanacetum vulgare, Simmondsia chinensis, Arabidopsis thaliana,Lotus japonicas, or Candida tropicalis. In exemplary embodiments, thealcohol oxidase comprises a sequence selected from GenBank AccessionNos. Q00922, F2QY27, Q6QIR6, Q8LDP0, and L7VFV2.

In alternative embodiments, the fatty alcohol may be converted into afatty aldehyde using chemical methods, including but not limited to, theuse of TEMPO-bleach, TEMPO-copper-air, TEMPO-PhI(OAc)₂, Swern oxidation,or noble metal-air.

As described above, in some embodiments, the recombinant microorganismaccording to the first or second aspect further comprises at least oneendogenous or exogenous nucleic acid molecule encoding an acetyltransferase capable of catalyzing the conversion of a C₆-C₂₄ fattyalcohol into a corresponding C₆-C₂₄ fatty acetate. In certainembodiments, the acetyl transferase, or the nucleic acid sequence thatencodes it, can be isolated from organisms of the species Saccharomycescerevisiae, Danaus plexippus, Heliotis virescens, Bombyx mori, Agrotisipsilon, Agrotis segetum, Euonymus alatus. In exemplary embodiments, theacetyl transferase comprises a sequence selected from GenBank AccessionNos. AY242066, AY242065, AY242064, AY242063, AY242062, EHJ65205,ACX53812, NP_001182381, EHJ65977, EHJ68573, KJ579226, GU594061.

In alternative embodiments, the fatty alcohol may be converted into afatty acetate using chemical methods, e.g., via chemical catalysisutilizing a chemical agent such as acetyl chloride, acetic anhydride,butyryl chloride, butyric anhydride, propanoyl chloride and propionicanhydride.

In some embodiments, the recombinant microorganism comprising abiosynthesis pathway for the production of an unsaturated C₆-C₂₄ fattyalcohol, aldehyde, or acetate may further be engineered to express oneor more nucleic acids encoding protein or polypeptide which, whenexpressed, is toxic to an insect. Exemplary toxicant producing genessuitable for the present disclosure can be obtained fromentomopathogenic organism, such as Bacillus thuringiensis, Pseudomonasaeruginosa, Serratia marcescens, and members of the genus Streptomyces.In an exemplary embodiment, the recombinant microorganism comprising abiosynthesis pathway for the production of an unsaturated C₆-C₂₄ fattyalcohol, aldehyde, or acetate may further be engineered to express anucleic acid encoding a Bacillus thuringiensis (“Bt”) toxin. Inadditional or alternative embodiments, the recombinant microorganismcomprising a biosynthesis pathway for the production of an unsaturatedC₆-C₂₄ fatty alcohol, aldehyde, or acetate may further be engineered toexpress a nucleic acid encoding other toxic proteins such as spidervenom.

In some embodiments, the recombinant microorganism comprising abiosynthesis pathway for the production of an unsaturated C₆-C₂₄ fattyalcohol, aldehyde, or acetate may further be engineered to express anRNAi molecule which, when expressed, produces an oligonucleotide that istoxic to an insect.

In some embodiments, the recombinant microorganism comprising abiosynthesis pathway for the production of an unsaturated C₆-C₂₄ fattyalcohol, aldehyde, or acetate may further be engineered to express ametabolic pathway which, when expressed, produces a small molecule thatis toxic to an insect. Non-limiting examples of toxic small moleculesinclude azadirachtin, spinosad, avermectin, pyrethrins, and variousterpenoids.

In various embodiments described herein, the recombinant microorganismcomprising a biosynthesis pathway for the production of an unsaturatedC₆-C₂₄ fatty alcohol, aldehyde, or acetate may be a eukaryoticmicroorganism, such as a yeast, a filamentous fungi, or an algae, oralternatively, a prokaryotic microorganism, such as a bacterium. Forinstance, suitable host cells can include cells of a genus selected fromthe group consisting of Yarrowia, Candida, Saccharomyces, Pichia,Hansenula, Clostridium, Zymomonas, Escherichia, Salmonella, Rhodococcus,Pseudomonas, Bacillus, Lactobacillus, Enterococcus, Alcaligenes,Klebsiella, Paenibacillus, Arthrobacter, Corynebacterium,Brevibacterium, and Streptomyces.

In some embodiments, the recombinant microorganism comprising abiosynthesis pathway for the production of an unsaturated C₆-C₂₄ fattyalcohol, aldehyde, or acetate is a yeast. Examples of suitable yeastsinclude yeasts of a genus selected from the group consisting ofYarrowia, Candida, Saccharomyces, Pichia, Hansenula, Kluyveromyces,Issatchenkia, Zygosaccharomyces, Debaryomyces, Schizosaccharomyces,Pachysolen, Cryptococcus, Trichosporon, Rhodotorula, or Myxozyma. Incertain embodiments, the yeast is an oleaginous yeast. Exemplaryoleaginous yeasts suitable for use in the present disclosure includemembers of the genera Yarrowia, Candida, Rhodotorula, Rhodosporidium,Cryptococcus, Trichosporon, and Lipomyces, including, but not limited tothe species of Yarrowia lipolytica, Candida tropicalis, Rhodosporidiumtoruloides, Lipomyces starkey, L. lipoferus, C. revkaufi, C.pulcherrima, C. utilis, Rhodotorula minuta, Trichosporon pullans, T.cutaneum, Cryptococcus curvatus, R. glutinis, and R. graminis.

As will be understood in the art, endogenous enzymes can convertcritical substrates and/or intermediates upstream of or within theunsaturated C₆-C₂₄ fatty alcohol, aldehyde, or acetate biosynthesispathway into unwanted by-products. Accordingly, in some embodiments, therecombinant microorganism is manipulated to delete, disrupt, mutate,and/or reduce the activity of one or more endogenous enzymes thatcatalyzes a reaction in a pathway that competes with the unsaturatedC₆-C₂₄ fatty alcohol, aldehyde, or acetate biosynthesis pathway.

In one embodiment, the recombinant microorganism is manipulated todelete, disrupt, mutate, and/or reduce the activity of one or moreendogenous enzymes that catalyzes the conversion of a fatty acid into aω-hydroxyfatty acid. In the context of a recombinant yeastmicroorganism, the recombinant yeast microorganism is engineered todelete, disrupt, mutate, and/or reduce the activity of one or moreenzyme selected from XP_504406, XP_504857, XP_504311, XP_500855,XP_500856, XP_500402, XP_500097, XP_501748, XP_500560, XP_501148,XP_501667, XP_500273, BAA02041, CAA39366, CAA39367, BAA02210, BAA02211,BAA02212, BAA02213, BAA02214, AA073952, AA073953, AA073954, AA073955,AA073956, AA073958, AA073959, AA073960, AA073961, AA073957,XP_002546278, or homologs thereof. In the context of a recombinantbacterial microorganism, the recombinant bacterial microorganism isengineered to delete, disrupt, mutate, and/or reduce the activity of oneor more enzyme selected from BAM49649, AAB80867, AAB17462, ADL27534,AAU24352, AAA87602, CAA34612, ABM17701, AAA25760, CAB51047, AAC82967,WP_011027348, or homologs thereof.

In another embodiment, the recombinant microorganism is manipulated todelete, disrupt, mutate, and/or reduce the activity of one or moreendogenous enzymes that catalyzes the conversion of a fatty acyl-CoAinto α,β-enoyl-CoA. In the context of a recombinant yeast microorganism,the recombinant yeast microorganism is engineered to delete, disrupt,mutate, and/or reduce the activity of one or more enzyme selected fromCAA04659, CAA04660, CAA04661, CAA04662, CAA04663, CAG79214, AAA34322,AAA34361, AAA34363, CAA29901, BAA04761, AAA34891, or homologs thereof.In the context of a recombinant bacterial microorganism, the recombinantbacterial microorganism is engineered to delete, disrupt, mutate, and/orreduce the activity of one or more enzyme selected from AAB08643,CAB15271, BAN55749, CAC44516, ADK16968, AEI37634, WP_000973047,WP_025433422, WP_035184107, WP_026484842, CEL80920, WP_026818657,WP_005293707, WP_005883960, or homologs thereof.

In embodiments where the recombinant microorganism is a yeastmicroorganism, the recombinant microorganism is manipulated to delete,disrupt, mutate, and/or reduce the activity of one or more enzymeinvolved in peroxisome assembly and/or peroxisome enzyme import. Therecombinant yeast microorganism is engineered to delete, disrupt,mutate, and/or reduce the activity of one or more enzyme selected fromXP_505754, XP_501986, XP_501311, XP_504845, XP_503326, XP_504029,XP_002549868, XP_002547156, XP_002545227, XP_002547350, XP_002546990,EIW11539, EIW08094, EIW11472, EIW09743, EIW08286, or homologs thereof.

In another embodiment, the recombinant microorganism is manipulated todelete, disrupt, mutate, and/or reduce the activity of one or moreendogenous reductase or desaturase enzymes that interferes with theunsaturated C₆-C₂₄ fatty alcohol, aldehyde, or acetate, i.e., catalyzesthe conversion of a pathway substrate or product into an unwantedby-product.

In another embodiment, the recombinant microorganism is manipulated todelete, disrupt, mutate, and/or reduce the activity of one or moreendogenous alcohol oxidase or alcohol dehydrogenase enzymes thatcatalyzes the unwanted conversion of the desired product, e.g.,unsaturated C₆-C₂₄ fatty alcohol into a corresponding unsaturated C₆-C₂₄fatty aldehyde.

In another embodiment, the recombinant microorganism is manipulated todelete, disrupt, mutate, and/or reduce the activity of one or moreendogenous enzymes that catalyzes a reaction in a pathway that competeswith the biosynthesis pathway for one or more unsaturated fatty acyl-CoAintermediates. In one embodiment, the one or more endogenous enzymescomprise one or more diacylglycerol acyltransferases. In the context ofa recombinant yeast microorganism, the recombinant yeast microorganismis engineered to delete, disrupt, mutate, and/or reduce the activity ofone or more diacylglycerol acyltransferases selected from the groupconsisting of YALI0E32769g, YALI0D07986g and CTRG 06209, or homologthereof. In another embodiment, the one or more endogenous enzymescomprise one or more glycerolphospholipid acyltransferases. In thecontext of a recombinant yeast microorganism, the recombinant yeastmicroorganism is engineered to delete, disrupt, mutate, and/or reducethe activity of one or more glycerolphospholipid acyltransferasesselected from the group consisting of YALI0E16797g and CTG_04390, orhomolog thereof. In another embodiment, the one or more endogenousenzymes comprise one or more acyl-CoA/sterol acyltransferases. In thecontext of a recombinant yeast microorganism, the recombinant yeastmicroorganism is engineered to delete, disrupt, mutate, and/or reducethe activity of one or more acyl-CoA/sterol acyltransferases selectedfrom the group consisting of YALI0F06578g, CTRG 01764 and CTRG 01765, orhomolog thereof.

In another embodiment, the recombinant microorganism is manipulated todelete, disrupt, mutate, and/or reduce the activity of one or moreendogenous enzymes that catalyzes a reaction in a pathway that oxidizesfatty aldehyde intermediates. In one embodiment, the one or moreendogenous enzymes comprise one or more fatty aldehyde dehydrogenases.In the context of a recombinant yeast microorganism, the recombinantyeast microorganism is engineered to delete, disrupt, mutate, and/orreduce the activity of one or more fatty aldehyde dehydrogenasesselected from the group consisting of YALI0A17875g, YALI0E15400g,YALI0B01298g, YALI0F23793g, CTRG 05010 and CTRG 04471, or homologthereof.

In another embodiment, the recombinant microorganism is manipulated todelete, disrupt, mutate, and/or reduce the activity of one or moreendogenous enzymes that catalyzes a reaction in a pathway that consumesfatty acetate products. In one embodiment, the one or more endogenousenzymes comprise one or more sterol esterases. In the context of arecombinant yeast microorganism, the recombinant yeast microorganism isengineered to delete, disrupt, mutate, and/or reduce the activity of oneor more sterol esterases selected from the group consisting ofYALI0E32035g, YALI0E00528g, CTRG 01138, CTRG 01683 and CTRG 04630, orhomolog thereof. In another embodiment, the one or more endogenousenzymes comprise one or more triacylglycerol lipases. In the context ofa recombinant yeast microorganism, the recombinant yeast microorganismis engineered to delete, disrupt, mutate, and/or reduce the activity ofone or more triacylglycerol lipases selected from the group consistingof YALI0D17534g, YALI0F10010g, CTRG 00057 and CTRG 06185, or homologthereof. In another embodiment, the one or more endogenous enzymescomprise one or more monoacylglycerol lipases. In the context of arecombinant yeast microorganism, the recombinant yeast microorganism isengineered to delete, disrupt, mutate, and/or reduce the activity of oneor more monoacylglycerol lipases selected from the group consisting ofYALI0C14520g, CTRG 03360 and CTRG 05049, or homolog thereof. In anotherembodiment, the one or more endogenous enzymes comprise one or moreextracellular lipases. In the context of a recombinant yeastmicroorganism, the recombinant yeast microorganism is engineered todelete, disrupt, mutate, and/or reduce the activity of one or moreextracellular lipases selected from the group consisting ofYALI0A20350g, YALI0D19184g, YALI0B09361g, CTRG 05930, CTRG 04188, CTRG02799, CTRG 03052 and CTRG 03885, or homolog thereof.

In embodiments where the recombinant microorganism is a yeastmicroorganism, one or more of the exogenous unsaturated C₆-C₂₄ fattyalcohol, aldehyde, or acetate pathway genes encodes an enzyme that islocalized to a yeast compartment selected from the group consisting ofthe cytosol, the mitochondria, or the endoplasmic reticulum. In anexemplary embodiment, one or more of the exogenous pathway genes encodesan enzyme that is localized to the endoplasmic reticulum. In anotherembodiment, at least two exogenous pathway genes encode an enzyme thatis localized to the endoplasmic reticulum. In yet another embodiment,all exogenous pathway genes encodes an enzyme that is localized to theendoplasmic reticulum.

In a fourth aspect, the present application provides methods ofproducing an unsaturated C₆-C₂₄ fatty alcohol, aldehyde, or acetateusing a recombinant microorganism as described herein. In oneembodiment, the method includes cultivating the recombinantmicroorganism in a culture medium containing a feedstock providing acarbon source until the unsaturated C₆-C₂₄ fatty alcohol, aldehyde, oracetate is produced and optionally, recovering the unsaturated C₆-C₂₄fatty alcohol, aldehyde, or acetate. Once produced, the unsaturatedC₆-C₂₄ fatty alcohol, aldehyde, or acetate may be isolated from thefermentation medium using various methods known in the art including,but not limited to, distillation, membrane-based separation gasstripping, solvent extraction, and expanded bed adsorption.

In some embodiments, the recombinant microorganism, e.g., a yeast, maybe recovered and produced in dry particulate form. In embodimentsinvolving yeast, the yeast may be dried to produce powdered yeast. Insome embodiments, the process for producing powdered yeast comprisesspray drying a liquid yeast composition in air, optionally followed byfurther drying. In some embodiments, the recombinant microorganismcomposition will comprise the unsaturated C₆-C₂₄ fatty alcohol,aldehyde, or acetate when dried.

As described herein, preferred recombinant microorganisms of thedisclosure will have the ability to utilize alkanes and fatty acids ascarbon sources. However, as will be understood in the art, a variety ofcarbon sources may be utilized, including but not limited to, varioussugars (e.g., glucose, fructose, or sucrose), glycerol, alcohols (e.g.,ethanol), organic acids, lignocellulose, proteins, carbon dioxide,carbon monoxide, as well as the aforementioned alkanes and fatty acids.In an exemplary embodiment, the recombinant microorganism will convertthe carbon source to the unsaturated C₆-C₂₄ fatty alcohol, aldehyde, oracetate under aerobic conditions.

As highlighted above, the present application provides methods ofproducing one or more unsaturated C₆-C₂₄ fatty alcohols, aldehydes, oracetates using a recombinant microorganism as described herein. In someembodiments, the product is an insect pheromone. As will be appreciatedby the skilled artisan equipped with the instant disclosure, a varietyof different exogenous and endogenous enzymes can be expressed in arecombinant host microorganism to produce a desired insect pheromone.Exemplary insect pheromones in the form of fatty alcohols, fattyaldehydes, or fatty acetates capable of being generated using therecombinant microorganisms and methods described herein include, but arenot limited to, (Z)-11-hexadecenal, (Z)-11hexadecenyl acetate,(Z)-9-tetradecenyl acetate, (Z,Z)-11,13-hexadecadienal,(9Z,11E)-hexadecadienal, (E,E)-8,10-dodecadien-1-ol,(7E,9Z)-dodecadienyl acetate, (Z)-3-nonen-1-ol, (Z)-5-decen-1-ol,(Z)-5-decenyl acetate, (E)-5-decen-1-ol, (E)-5-decenyl acetate,(Z)-7-dodecen-1-ol, (Z)-7-dodecenyl acetate, (E)-8-dodecen-1-ol,(E)-8-dodecenyl acetate, (Z)-8-dodecen-1-ol, (Z)-8-dodecenyl acetate,(Z)-9-dodecen-1-ol, (Z)-9-dodecenyl acetate, (Z)-9-tetradecen-1-ol,(Z)-11-tetraceden-1-ol, (Z)-11-tetracedenyl acetate,(E)-11-tetradecen-1-ol, (E)-11-tetradecenyl acetate,(Z)-7-hexadecen-1-ol, (Z)-7-hexadecenal, (Z)-9-hexadecen-1-ol,(Z)-9-hexadecenal, (Z)-9-hexadecenyl acetate, (Z)-11-hexadecen-1-ol,(Z)-13-octadecen-1-ol, and (Z)-13-octadecenal.

In a fifth aspect, the present application provides compositionscomprising one of more of the insect pheromone-producing recombinantmicroorganisms described herein. In certain embodiments, the compositionmay further comprise one or more insect pheromones produced by therecombinant microorganism. In further embodiments, the may additionallycomprise one or more toxic proteins or polypeptides produced by therecombinant microorganism.

BRIEF DESCRIPTION OF DRAWINGS

Illustrative embodiments of the disclosure are illustrated in thedrawings, in which:

FIG. 1 illustrates the conversion of a saturated fatty acyl-CoA to anunsaturated fatty alcohol.

FIG. 2 illustrates the conversion of a saturated fatty acid to a mono-or poly-unsaturated fatty aldehyde, alcohol, or acetate.

FIG. 3 illustrates an additional pathway for the conversion of asaturated fatty acid to a mono- or poly-unsaturated fatty aldehyde,alcohol, or acetate

FIG. 4 illustrates a pathway for the conversion of a saturated fattyacid to various trienes, dienes, epoxides, and odd-numbered pheromones.

FIG. 5 shows Z11-hexadecenol production from W303A and BY4742 ΔPOX1.Strain expressing empty vector (EV), S. littoralis reductase (FAR-SL),H. armigera reductase (FAR-HA), A. segetum reductase (FAR-AS). Errorbars represent standard deviation derived from N=2biologically-independent samples.

FIG. 6A-FIG. 6B shows sample chromatograms of biotransformation productof Z11-hexadecenoic acid using S. cerevisiae expressing either an emptyvector (FIG. 6A), or Helicoverpa armigera alcohol-forming reductase(FIG. 6B). Black lines: no substrate added. Purple line:Z11-hexadecenoic acid was added as substrate.

FIG. 7A-FIG. 7B shows a comparison of GC-MS fragmentation pattern ofZ11-hexadecenol authentic compound (FIG. 7A), and Z11-hexadecenolbiologically derived (FIG. 7B).

FIG. 8 shows biomass at the time of harvesting for product analysis ofW303A (wild type) and BY4742 ΔPOX1 (beta-oxidation deletion mutant).Strain expressing empty vector (EV), S. littoralis reductase (FAR-SL),H. armigera reductase (FAR-HA), A. segetum reductase (FAR-AS). Errorbars represent standard deviation derived from N=2biologically-independent samples.

FIG. 9 shows a Z11-hexedecenol calibration curve constructed using anauthentic standard. The samples were generated with the extraction andanalysis method described in Materials and Methods of Example 3. Errorbars represent standard deviation derived from N=3 samples.

FIG. 10 shows a pOLE1 cassette comprising an extended OLE1 promotersequence (light yellow), OLE1 promoter (orange), OLE1 leader sequence(dark grey), a synthon such as an insect desaturase sequence (lightgrey), and the VSP13 terminator sequence (blue).

FIG. 11A-FIG. 11E shows validation of the pOLE1 cassette, andcomplementation assay. FIG. 11A: YPD+palmitoleic acid; FIG. 11B:YPD—palmitoleic acid; FIG. 11C: CM-Ura glucose+palmitoleic acid; FIG.11D: CM-Ura glucose—palmitoleic acid; FIG. 11E: Map of strains in FIG.11A-FIG. 11D. Dasher=GFP synthon.

FIG. 12A shows complementation of ΔOLE1 growth without UFA on YPD.

FIG. 12B shows complementation of ΔOLE1 growth without UFA on CM-Uraglucose.

FIG. 13A shows the full fatty acid spectrum of a ΔOLE1 strainexpressing: S. cerevisiae OLE1 desaturase (blue), chimeric T. nidesaturase (red).

FIG. 13B shows a focused fatty acid spectrum within 5.5-min-8-minretention time of S. cerevisiae ΔOLE1 strain expressing S. cerevisiaeOLE1 desaturase (red) and chimeric T. ni desaturase (blue).

FIG. 14A-FIG. 14B shows a comparison of GC-MS fragmentation pattern of(Z)-11-hexadecenoic acid from an authentic compound (FIG. 14A) andbiologically derived (FIG. 14B).

FIG. 15 shows C16 fatty alcohol production from ΔOLE1 expressing variousfatty alcohol pathway variants in culture supplemented with palmitic andpalmitoleic acid. Error bars represent 5% uncertainty of metabolitequantification accuracy.

FIG. 16 shows representative chromatograms of biotransformation productC16 fatty acids using S. cerevisiae expressing fatty alcohol pathwaysTN_desat-HA_reduc when fed with palmitic acid (black) and when fed withpalmitic and palmitoleic acids (orange). Profile of a negative controlstrain (harboring an empty vector) fed with palmitic acid (purple).

FIG. 17 shows that (Z)-11-hexadecenoic acid was detected in the cellpellets of S. cerevisiae expressing fatty alcohol pathwaysTN_desat-SL_reduc (blue), SC_desat-HA_reduc (red), TN_desat-HA_reduc(green), SC_desat-SL_reduc (pink).

FIG. 18 shows C16 fatty alcohol production from ΔOLE1 expressing variousfatty alcohol pathway variants in culture supplemented with palmiticacid only. Error bars represent 5% uncertainty of metabolitequantification accuracy.

FIG. 19A-FIG. 19C shows detection of (Z)-11-hexadecenol. FIG. 19A:Fragmentation pattern of an authentic standard. The m/z 297.3 was usedin follow up experiments to selectively detect the alcohol. To alsodetect the internal standard, the masses 208 and 387.3 were includedtoo. FIG. 19B: In addition to the detection of the specific massfragment, the retention time was used as second stage confirmation. Theretention time is 6.22. FIG. 19C: Comparison of the two differentregioisomers 9Z- and 11Z-hexadecenol when detected in SIM mode (297.3)with the same method.

FIG. 20 shows pXICL expression cassette architecture. The C. albicansOLE1 leader-A. segetum desaturase fusion is also shown.

FIG. 21A-FIG. 21D shows mCherry control integration. FIG. 21A: Negative(water-only) control transformation plate. FIG. 21B: pPV0137 mCherrytransformation plate. FIG. 21C: Patch plates from negative controlclones. FIG. 21D: Patch plates from pPV0137 clones.

FIG. 22 shows integration efficiency as a function of total observedcolonies. A control plate with no DNA added to the transformation wasobserved to have 350 colonies (indicated by orange line). The fractionof clones confirmed to be positive integrants is positively correlatedwith total colony count. A sharp increase is observed above 6,000 totalcolonies. The data suggests that the presence of positive integrantsincreases the observed background growth. For some transformations theefficiency was high enough that the background population was smallrelative to the positive integrant population.

FIG. 23 shows a chromatogram overlay of Candida tropicalis SPV053strains. Compared to the mCherry (red) control experiment a clear peakat 6.22 min is observable for the A. transitella (blue) and H. zea(green) desaturase. Therefore, the formation of Z-11-hexadecenoic acidis only observable in strains expressing an active Z11-desaturase.

FIG. 24A-FIG. 24E shows confirmation of the 11Z-regioisomer. FIG. 24A:The specific peak with an ion fragment of 245 m/z was only observed inC. tropicalis SPV053 expressing either the Z11-desaturase from A.transitella or H. zea. FIG. 24B: The fragmentation patterns of theauthentic standard. FIG. 24D: The fragmentation patterns of the newlyformed compound in samples with expressed desaturase from H. zea matchthose of the standard. FIG. 24E: The fragmentation patterns of the newlyformed compound in samples with expressed desaturase from A. transitellamatch those of the standard. FIG. 24C: The fragmentation patterns of themCherry control significantly differ from those of FIG. 24B, FIG. 24Dand FIG. 24E.

FIG. 25A-FIG. 25B shows a GC-FID chromatogram of different C. tropicalisSPV053 strains incubated with methyl tretradecanoate. FIG. 25A: Overallspectrum. The occurrence of the Z11-C16:1 peak is observable for thestrains expressing the Z11-desaturases from A. transitella and H. zea.FIG. 25B: Zoom of the C14 to C18 area. A new peak is visible at 4.8 min,which could correspond to Z11-C14:1. Another peak near Z9-C18:1 is alsovisible, which could correspond to Z11-C18:1.

FIG. 26 shows only codon optimized H. zea desaturase variants producedetectable Z11-hexadecenoic acid in SPV140 screen. control=pPV101integrants of SPV140, T. ni native=T. ni Z11 desaturase with nativecodon usage (pPV195), T. ni HS opt=T. ni Z11 desaturase with Homosapiens codon optimization (pPV196), T. ni HS opt Yl leader=T. ni Z11desaturase with Homo sapiens codon optimization and swapped Y.lipolytica OLE1 leader sequence (pPV197), H. zea native=H. zea Z11desaturase with native codon usage (pPV198), H. zea HS opt=H. zea Z11desaturase with Homo sapiens codon optimization (pPV199), H. zea HS optYl leader=H. zea Z11 desaturase with Homo sapiens codon optimization andswapped Y. lipolytica OLE1 leader sequence (pPV200), A. transitellanative A. transitella Z11 desaturase with native codon usage (pPV201).All data average of 3 biological replicates. Error bars representstandard deviation.

FIG. 27 shows only codon optimized H. zea desaturase variants producedetectable Z11-hexadecenoic acid in SPV300 screen. Labels indicateparent strain and plasmid of desaturase expression cassette.pPV101=hrGFP control, pPV198=H. zea Z11 desaturase with native codonusage, pPV199=H. zea Z11 desaturase with Homo sapiens codonoptimization, pPV200=H. zea Z11 desaturase with Homo sapiens codonoptimization and swapped Y. lipolytica OLE1 leader sequence, pPV201=A.transitella Z11 desaturase with native codon usage.

FIG. 28 shows final cell densities for desaturase screen in SPV140 andSPV300 backgrounds. SPV300 strains with integrated desaturase cassettesgrew to higher cell densities.

FIG. 29 shows individual isolate Z11-hexadecenoic acid titers for SPV140and SPV300 strains expressing H. zea Z11 desaturase with H. sapienscodon optimization.

FIG. 30 shows a chromatogram overlay of extracted metabolites forZ11-16OH producing strain (SPV0490) versus control strain (SPV0488) ofCandida viswanathii (tropicalis).

FIG. 31 illustrates pathways that can be deleted or disrupted to reduceor eliminate competition with the biosynthesis pathway for theproduction of a mono- or poly-unsaturated C₆-C₂₄ fatty alcohol,aldehyde, or acetate.

FIG. 32A-FIG. 32B shows Z9-16OH and Z11-16OH titers in YPD (FIG. 32A)and Semi-Defined C:N=80 (FIG. 32B) media for pEXP clones. Ten isolatesexpressing the H. zea desaturase under the TEF promoter and H. armigerareductase under the EXP promoter from two independent competent cellpreparations (Comp. Cell Preparation 1, Comp. Cell Preparation 2) werecompared to a parental negative control (SPV300) and a desaturase onlynegative control (SPV459 Hz_desat only). Error bars represent the SEM(standard error of the mean) measured from technical replicates for eachstrain and condition (N=2). *One replicate from Clone 5 and Clone 18under the Semi-Defined C:N=80 condition was lost during sample work-upso the titers for that condition are from a single data point (N=1,Comp. Cell Preparation 1 Clone 18 and Comp. Cell Preparation 2 Clone 5).

FIG. 33A-FIG. 33B shows profiles of 16-carbon fatty acid species in YPD(FIG. 33A) and Semi-Defined C:N=80 (FIG. 33B) media for pEXP1 clones.The 16-carbon lipid profiles of 5 select clones expressing the H. zeadesaturase under the TEF promter and H. armigera reductase under the EXPpromoter are compared to a parental negative control (SPV300) and adesaturase only negative control (SPV459 Hz_desat only). Error barsrepresent the SEM (standard error of the mean) measured from technicalreplicates for each strain and condition (N=2).

FIG. 34 shows Z9-16OH and Z11-16OH titers in Semi-Defined C:N=80 mediafor pTAL1 clones. Nine isolates expressing the H. zea desaturase underthe TEF promoter and H. armigera reductase under the TAL promoter werecompared to a parental negative control (SPV300) and positive Bdrpathway controls using the EXP promoter to drive H. armigera FARexpression (SPV575, SPV578). Error bars represent the SEM (standarderror of the mean) measured from technical replicates for each strainand condition (N=2).

FIG. 35 shows profiles of 16-carbon fatty acid species in Semi-DefinedC:N=80 medium for pTAL1 clones. The 16-carbon lipid profiles of 5 selectclones expressing the H. zea desaturase under the TEF promoter and H.armigera reductase under the EXP promoter are compared to a parentalnegative control (SPV300) and positive Bdr pathway controls using theEXP promoter to drive H. armigera FAR expression (SPV575, SPV578). Errorbars represent the SEM (standard error of the mean) measured fromtechnical replicates for each strain and condition (N=2). * indicatesclones for which one of the replicates was lost during sampleprocessing, N=1.

FIG. 36 shows full Bdr pathway pTAL1 screen (strains expressing H. zeaZ11 desaturase (pTEF) and H. armigera FAR) full lipid profiles inSemi-Defined C:N=80 medium after 48 hours of bioconversion. Error barsrepresent the SEM (standard error of the mean) measured from technicalreplicates for each strain and condition (N=2).* indicates clones forwhich one of the replicates was lost during sample processing, N=1.

FIG. 37A-FIG. 37B shows SPV471 (H222 APAAAF expressing native Y.lipolytica OLE1 and H. armigera FAR) Z9-16OH (FIG. 37A) and fatty acid(FIG. 37B) titers in Semi-Defined C:N=80 medium after 24 hours ofbioconversion. Error bars represent the SEM (standard error of the mean)measured from technical replicates for each strain and condition (N=2).

FIG. 38 shows SPV471 (H222 APAAAF expressing native Y. lipolytica OLE1and H. armigera FAR) full lipid profiles in Semi-Defined C:N=80 mediumafter 24 hours of bioconversion.

FIG. 39A-FIG. 39B shows SPV471 (H222 APAAAF expressing native Y.lipolytica OLE1 and H. armigera FAR) Z9-16OH (FIG. 39A) and Z9-16Acid(FIG. 39B) titer time courses. Bioconversion of 16Acid was conducted inSemi-Defined C:N=80 medium using a methyl palmitate (16Acid) substrate.

FIG. 40 shows a scheme illustrating exemplary means by which therecombinant microorganisms disclosed herein can be used to createsynthetic blends of insect pheromones.

SEQUENCES

A sequence listing for SEQ ID NO: 1-SEQ ID NO: 39 is part of thisapplication and is incorporated by reference herein. The sequencelisting for SEQ ID NO: 1-SEQ ID NO: 38 is provided at the end of thisdocument.

DETAILED DESCRIPTION Definitions

The following definitions and abbreviations are to be used for theinterpretation of the disclosure.

As used herein and in the appended claims, the singular forms “a,” “an,”and “the” include plural referents unless the context clearly dictatesotherwise. Thus, for example, reference to “a pheromone” includes aplurality of such pheromones and reference to “the microorganism”includes reference to one or more microorganisms, and so forth.

As used herein, the terms “comprises,” “comprising,” “includes,”“including,” “has,” “having, “contains,” “containing,” or any othervariation thereof, are intended to cover a non-exclusive inclusion. Acomposition, mixture, process, method, article, or apparatus thatcomprises a list of elements is not necessarily limited to only thoseelements but may include other elements not expressly listed or inherentto such composition, mixture, process, method, article, or apparatus.Further, unless expressly stated to the contrary, “or” refers to aninclusive “or” and not to an exclusive “or.”

The terms “about” and “around,” as used herein to modify a numericalvalue, indicate a close range surrounding that explicit value. If “X”were the value, “about X” or “around X” would indicate a value from 0.9Xto 1.1X, or, in some embodiments, a value from 0.95X to 1.05X. Anyreference to “about X” or “around X” specifically indicates at least thevalues X, 0.95X, 0.96X, 0.97X, 0.98X, 0.99X, 1.01X, 1.02X, 1.03X, 1.04X,and 1.05X. Thus, “about X” and “around X” are intended to teach andprovide written description support for a claim limitation of, e.g.,“0.98X.”

As used herein, the terms “microbial,” “microbial organism,” and“microorganism” include any organism that exists as a microscopic cellthat is included within the domains of archaea, bacteria or eukarya, thelatter including yeast and filamentous fungi, protozoa, algae, or higherProtista. Therefore, the term is intended to encompass prokaryotic oreukaryotic cells or organisms having a microscopic size and includesbacteria, archaea, and eubacteria of all species as well as eukaryoticmicroorganisms such as yeast and fungi. Also included are cell culturesof any species that can be cultured for the production of a chemical.

As described herein, in some embodiments, the recombinant microorganismsare prokaryotic microorganism. In some embodiments, the prokaryoticmicroorganisms are bacteria. “Bacteria”, or “eubacteria”, refers to adomain of prokaryotic organisms. Bacteria include at least elevendistinct groups as follows: (1) Gram-positive (gram+) bacteria, of whichthere are two major subdivisions: (1) high G+C group (Actinomycetes,Mycobacteria, Micrococcus, others) (2) low G+C group (Bacillus,Clostridia, Lactobacillus, Staphylococci, Streptococci, Mycoplasmas);(2) Proteobacteria, e.g., Purple photosynthetic+non-photosyntheticGram-negative bacteria (includes most “common” Gram-negative bacteria);(3) Cyanobacteria, e.g., oxygenic phototrophs; (4) Spirochetes andrelated species; (5) Planctomyces; (6) Bacteroides, Flavobacteria; (7)Chlamydia; (8) Green sulfur bacteria; (9) Green non-sulfur bacteria(also anaerobic phototrophs); (10) Radioresistant micrococci andrelatives; (11) Thermotoga and Thermosipho thermophiles.

“Gram-negative bacteria” include cocci, nonenteric rods, and entericrods. The genera of Gram-negative bacteria include, for example,Neisseria, Spirillum, Pasteurella, Brucella, Yersinia, Francisella,Haemophilus, Bordetella, Escherichia, Salmonella, Shigella, Klebsiella,Proteus, Vibrio, Pseudomonas, Bacteroides, Acetobacter, Aerobacter,Agrobacterium, Azotobacter, Spirilla, Serratia, Vibrio, Rhizobium,Chlamydia, Rickettsia, Treponema, and Fusobacterium.

“Gram positive bacteria” include cocci, nonsporulating rods, andsporulating rods. The genera of gram positive bacteria include, forexample, Actinomyces, Bacillus, Clostridium, Corynebacterium,Erysipelothrix, Lactobacillus, Listeria, Mycobacterium, Myxococcus,Nocardia, Staphylococcus, Streptococcus, and Streptomyces.

The term “recombinant microorganism” and “recombinant host cell” areused interchangeably herein and refer to microorganisms that have beengenetically modified to express or to overexpress endogenous enzymes, toexpress heterologous enzymes, such as those included in a vector, in anintegration construct, or which have an alteration in expression of anendogenous gene. By “alteration” it is meant that the expression of thegene, or level of a RNA molecule or equivalent RNA molecules encodingone or more polypeptides or polypeptide subunits, or activity of one ormore polypeptides or polypeptide subunits is up regulated or downregulated, such that expression, level, or activity is greater than orless than that observed in the absence of the alteration. For example,the term “alter” can mean “inhibit,” but the use of the word “alter” isnot limited to this definition. It is understood that the terms“recombinant microorganism” and “recombinant host cell” refer not onlyto the particular recombinant microorganism but to the progeny orpotential progeny of such a microorganism. Because certain modificationsmay occur in succeeding generations due to either mutation orenvironmental influences, such progeny may not, in fact, be identical tothe parent cell, but are still included within the scope of the term asused herein.

The term “expression” with respect to a gene sequence refers totranscription of the gene and, as appropriate, translation of theresulting mRNA transcript to a protein. Thus, as will be clear from thecontext, expression of a protein results from transcription andtranslation of the open reading frame sequence. The level of expressionof a desired product in a host cell may be determined on the basis ofeither the amount of corresponding mRNA that is present in the cell, orthe amount of the desired product encoded by the selected sequence. Forexample, mRNA transcribed from a selected sequence can be quantitated byqRT-PCR or by Northern hybridization (see Sambrook et al., MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press(1989)). Protein encoded by a selected sequence can be quantitated byvarious methods, e.g., by ELISA, by assaying for the biological activityof the protein, or by employing assays that are independent of suchactivity, such as western blotting or radioimmunoassay, using antibodiesthat recognize and bind the protein. See Sambrook et al., 1989, supra.

The term “polynucleotide” is used herein interchangeably with the term“nucleic acid” and refers to an organic polymer composed of two or moremonomers including nucleotides, nucleosides or analogs thereof,including but not limited to single stranded or double stranded, senseor antisense deoxyribonucleic acid (DNA) of any length and, whereappropriate, single stranded or double stranded, sense or antisenseribonucleic acid (RNA) of any length, including siRNA. The term“nucleotide” refers to any of several compounds that consist of a riboseor deoxyribose sugar joined to a purine or a pyrimidine base and to aphosphate group, and that are the basic structural units of nucleicacids. The term “nucleoside” refers to a compound (as guanosine oradenosine) that consists of a purine or pyrimidine base combined withdeoxyribose or ribose and is found especially in nucleic acids. The term“nucleotide analog” or “nucleoside analog” refers, respectively, to anucleotide or nucleoside in which one or more individual atoms have beenreplaced with a different atom or with a different functional group.Accordingly, the term polynucleotide includes nucleic acids of anylength, DNA, RNA, analogs and fragments thereof. A polynucleotide ofthree or more nucleotides is also called nucleotidic oligomer oroligonucleotide.

It is understood that the polynucleotides described herein include“genes” and that the nucleic acid molecules described herein include“vectors” or “plasmids.” Accordingly, the term “gene”, also called a“structural gene” refers to a polynucleotide that codes for a particularsequence of amino acids, which comprise all or part of one or moreproteins or enzymes, and may include regulatory (non-transcribed) DNAsequences, such as promoter sequences, which determine for example theconditions under which the gene is expressed. The transcribed region ofthe gene may include untranslated regions, including introns,5′-untranslated region (UTR), and 3′-UTR, as well as the codingsequence.

The term “enzyme” as used herein refers to any substance that catalyzesor promotes one or more chemical or biochemical reactions, which usuallyincludes enzymes totally or partially composed of a polypeptide orpolypeptides, but can include enzymes composed of a different moleculeincluding polynucleotides.

As used herein, the term “non-naturally occurring,” when used inreference to a microorganism organism or enzyme activity of thedisclosure, is intended to mean that the microorganism organism orenzyme has at least one genetic alteration not normally found in anaturally occurring strain of the referenced species, includingwild-type strains of the referenced species. Genetic alterationsinclude, for example, modifications introducing expressible nucleicacids encoding metabolic polypeptides, other nucleic acid additions,nucleic acid deletions and/or other functional disruption of themicroorganism's genetic material. Such modifications include, forexample, coding regions and functional fragments thereof, forheterologous, homologous, or both heterologous and homologouspolypeptides for the referenced species. Additional modificationsinclude, for example, non-coding regulatory regions in which themodifications alter expression of a gene or operon. Exemplarynon-naturally occurring microorganism or enzyme activity includes thehydroxylation activity described above.

The term “exogenous” as used herein with reference to various molecules,e.g., polynucleotides, polypeptides, enzymes, etc., refers to moleculesthat are not normally or naturally found in and/or produced by a givenyeast, bacterium, organism, microorganism, or cell in nature.

On the other hand, the term “endogenous” or “native” as used herein withreference to various molecules, e.g., polynucleotides, polypeptides,enzymes, etc., refers to molecules that are normally or naturally foundin and/or produced by a given yeast, bacterium, organism, microorganism,or cell in nature.

The term “heterologous” as used herein in the context of a modified hostcell refers to various molecules, e.g., polynucleotides, polypeptides,enzymes, etc., wherein at least one of the following is true: (a) themolecule(s) is/are foreign (“exogenous”) to (i.e., not naturally foundin) the host cell; (b) the molecule(s) is/are naturally found in (e.g.,is “endogenous to”) a given host microorganism or host cell but iseither produced in an unnatural location or in an unnatural amount inthe cell; and/or (c) the molecule(s) differ(s) in nucleotide or aminoacid sequence from the endogenous nucleotide or amino acid sequence(s)such that the molecule differing in nucleotide or amino acid sequencefrom the endogenous nucleotide or amino acid as found endogenously isproduced in an unnatural (e.g., greater than naturally found) amount inthe cell.

The term “homolog,” as used herein with respect to an original enzyme orgene of a first family or species, refers to distinct enzymes or genesof a second family or species which are determined by functional,structural, or genomic analyses to be an enzyme or gene of the secondfamily or species which corresponds to the original enzyme or gene ofthe first family or species. Homologs most often have functional,structural, or genomic similarities. Techniques are known by whichhomologs of an enzyme or gene can readily be cloned using genetic probesand PCR. Identity of cloned sequences as homologs can be confirmed usingfunctional assays and/or by genomic mapping of the genes.

A protein has “homology” or is “homologous” to a second protein if theamino acid sequence encoded by a gene has a similar amino acid sequenceto that of the second gene. Alternatively, a protein has homology to asecond protein if the two proteins have “similar” amino acid sequences.Thus, the term “homologous proteins” is intended to mean that the twoproteins have similar amino acid sequences. In certain instances, thehomology between two proteins is indicative of its shared ancestry,related by evolution.

The term “fatty acid” as used herein refers to a compound of structureR—COOH, wherein R is a C₆ to C₂₄ saturated, unsaturated, linear,branched or cyclic hydrocarbon and the carboxyl group is at position 1.In a particular embodiment, R is a C₆ to C₂₄ saturated or unsaturatedlinear hydrocarbon and the carboxyl group is at position 1.

The term “fatty alcohol” as used herein refers to an aliphatic alcoholhaving the formula R—OH, wherein R is a C₆ to C₂₄ saturated,unsaturated, linear, branched or cyclic hydrocarbon. In a particularembodiment, R is a C₆ to C₂₄ saturated or unsaturated linearhydrocarbon.

The term “fatty acyl-CoA” refers to a compound having the structureR—(CO)—S—R₁, wherein R₁ is Coenzyme A, and the term “fatty acyl-ACP”refers to a compound having the structure R—(CO)—S—R₁, wherein R₁ is anacyl carrier protein ACP.

Introduction

The present disclosure addresses the need for novel technologies for thecost-efficient production of valuable products from low-cost feedstocks.Specifically, the present inventors have addressed this need with thedevelopment of recombinant microorganisms capable of producing awide-range of unsaturated C₆-C₂₄ fatty alcohols, aldehydes, and acetatesincluding synthetic insect pheromones, fragrances, flavors, and polymerintermediates from low-cost feedstocks. Thus, aspects of the disclosureare based on the inventors' discovery that recombinant microorganismscan be engineered in order to produce valuable products from low-costfeedstocks, which circumvents conventional synthetic methodologies toproduce valuable products.

As discussed above, recombinant microorganisms can be engineered tosynthesize mono- or poly-unsaturated C₆-C₂₄ fatty alcohols. Mono- orpoly-unsaturated C₆-C₂₄ fatty alcohols synthesized as described hereincan be further converted into the corresponding aldehydes or acetates.Thus, various embodiments of the present disclosure can be used tosynthesize a variety of insect pheromones selected from fatty alcohols,aldehydes, and acetates. Additionally, embodiments described herein canalso be used for the synthesis of fragrances, flavors, and polymerintermediates.

Pheromones

As described above, embodiments of the disclosure provide for thesynthesis of one or more insect pheromones using a recombinantmicroorganism. A pheromone is a volatile chemical compound that issecreted by a particular insect for the function of chemicalcommunication within the species. That is, a pheromone is secreted orexcreted chemical factor that triggers a social response in members ofthe same species. There are, inter alia, alarm pheromones, food trailpheromones, sex pheromones, aggregation pheromones, epideicticpheromones, releaser pheromones, primer pheromones, and territorialpheromones, that affect behavior or physiology.

Non-limiting examples of insect pheromones which can be synthesizedusing the recombinant microorganisms and methods disclosed hereininclude linear alcohols, aldehydes, and acetates listed in Table 1.

TABLE 1   C₆-C₂₀ Linear Pheromones Name (E)-2-Decen-1-ol (E)-2-Decenylacetate (E)-2-Decenal (Z)-2-Decen-1-ol (Z)-2-Decenyl acetate(Z)-2-Decenal (E)-3-Decen-1-ol (Z)-3-Decenyl acetate (Z)-3-Decen-1-ol(Z)-4-Decen-1-ol (E)-4-Decenyl acetate (Z)-4-Decenyl acetate(Z)-4-Decenal (E)-5-Decen-1-ol (E)-5-Decenyl acetate (Z)-5-Decen-1-ol(Z)-5-Decenyl acetate (Z)-5-Decenal (E)-7-Decenyl acetate (Z)-7-Decenylacetate (E)-8-Decen-1-ol (E,E)-2,4-Decadienal (E,Z)-2,4-Decadienal(Z,Z)-2,4-Decadienal (E,E)-3,5-Decadienyl acetate (Z,E)-3,5-Decadienylacetate (Z,Z)-4,7-Decadien-1-ol (Z,Z)-4,7-Decadienyl acetate(E)-2-Undecenyl acetate (E)-2-Undecenal (Z)-5-Undecenyl acetate(Z)-7-Undecenyl acetate (Z)-8-Undecenyl acetate (Z)-9-Undecenyl acetate(E)-2-Dodecenal (Z)-3-Dodecen-1-ol (E)-3-Dodecenyl acetate(Z)-3-Dodecenyl acetate (E)-4-Dodecenyl acetate (E)-5-Dodecen-1-ol(E)-5-Dodecenyl acetate (Z)-5-Dodecen-1-ol (Z)-5-Dodecenyl acetate(Z)-5-Dodecenal (E)-6-Dodecen-1-ol (Z)-6-Dodecenyl acetate(E)-6-Dodecenal (E)-7-Dodecen-1-ol (E)-7-Dodecenyl acetate(E)-7-Dodecenal (Z)-7-Dodecen-1-ol (Z)-7-Dodecenyl acetate(Z)-7-Dodecenal (E)-8-Dodecen-1-ol (E)-8-Dodecenyl acetate(E)-8-Dodecenal (Z)-8-Dodecen-1-ol (Z)-8-Dodecenyl acetate(E)-9-Dodecen-1-ol (E)-9-Dodecenyl acetate (E)-9-Dodecenal(Z)-9-Dodecen-1-ol (Z)-9-Dodecenyl acetate (Z)-9-Dodecenal(E)-10-Dodecen-1-ol (E)-10-Dodecenyl acetate (E)-10-Dodecenal(Z)-10-Dodecen-1-ol (Z)-10-Dodecenyl acetate (E,Z)-3,5-Dodecadienylacetate (Z,E)-3,5-Dodecadienyl acetate (Z,Z)-3,6-Dodecadien-1-ol(E,E)-4,10-Dodecadienyl acetate (E,E)-5,7-Dodecadien-1-ol(E,E)-5,7-Dodecadienyl acetate (E,Z)-5,7-Dodecadien-1-ol(E,Z)-5,7-Dodecadienyl acetate (E,Z)-5,7-Dodecadienal(Z,E)-5,7-Dodecadien-1-ol (Z,E)-5,7-Dodecadienyl acetate(Z,E)-5,7-Dodecadienal (Z,Z)-5,7-Dodecadienyl acetate(Z,Z)-5,7-Dodecadienal (E,E)-7,9-Dodecadienyl acetate(E,Z)-7,9-Dodecadien-1-ol (E,Z)-7,9-Dodecadienyl acetate(E,Z)-7,9-Dodecadienal (Z,E)-7,9-Dodecadien-1-ol (Z,E)-7,9-Dodecadienylacetate (Z,Z)-7,9-Dodecadien-1-ol (Z,Z)-7,9-Dodecadienyl acetate(E,E)-8,10-Dodecadien-1-ol (E,E)-8,10-Dodecadienyl acetate(E,E)-8,10-Dodecadienal (E,Z)-8,10-Dodecadien-1-ol(E,Z)-8,10-Dodecadienyl acetate (E,Z)-8,10-Dodecadienal(Z,E)-8,10-Dodecadien-1-ol (Z,E)-8,10-Dodecadienyl acetate(Z,E)-8,10-Dodecadienal (Z,Z)-8,10-Dodecadien-1-ol(Z,Z)-8,10-Dodecadienyl acetate (Z,E,E)-3,6,8-Dodecatrien-1-ol(Z,Z,E)-3,6,8-Dodecatrien-1-ol (E)-2-Tridecenyl acetate (Z)-2-Tridecenylacetate (E)-3-Tridecenyl acetate (E)-4-Tridecenyl acetate(Z)-4-Tridecenyl acetate (Z)-4-Tridecenal (E)-6-Tridecenyl acetate(Z)-7-Tridecenyl acetate (E)-8-Tridecenyl acetate (Z)-8-Tridecenylacetate (E)-9-Tridecenyl acetate (Z)-9-Tridecenyl acetate(Z)-10-Tridecenyl acetate (E)-11-Tridecenyl acetate (Z)-11-Tridecenylacetate (E,Z)-4,7-Tridecadienyl acetate (Z,Z)-4,7-Tridecadien-1-ol(Z,Z)-4,7-Tridecadienyl acetate (E,Z)-5,9-Tridecadienyl acetate(Z,E)-5,9-Tridecadienyl acetate (Z,Z)-5,9-Tridecadienyl acetate(Z,Z)-7,11-Tridecadienyl acetate (E,Z,Z)-4,7,10-Tridecatrienyl acetate(E)-3-Tetradecen-1-ol (E)-3-Tetradecenyl acetate (Z)-3-Tetradecen-1-ol(Z)-3-Tetradecenyl acetate (E)-5-Tetradecen-1-ol (E)-5-Tetradecenylacetate (E)-5-Tetradecenal (Z)-5-Tetradecen-1-ol (Z)-5-Tetradecenylacetate (Z)-5-Tetradecenal (E)-6-Tetradecenyl acetate (Z)-6-Tetradecenylacetate (E)-7-Tetradecen-1-ol (E)-7-Tetradecenyl acetate(Z)-7-Tetradecen-1-ol (Z)-7-Tetradecenyl acetate (Z)-7-Tetradecenal(E)-8-Tetradecenyl acetate (Z)-8-Tetradecen-1-ol (Z)-8-Tetradecenylacetate (Z)-8-Tetradecenal (E)-9-Tetradecen-1-ol (E)-9-Tetradecenylacetate (Z)-9-Tetradecen-1-ol (Z)-9-Tetradecenyl acetate(Z)-9-Tetradecenal (E)-10-Tetradecenyl acetate (Z)-10-Tetradecenylacetate (E)-11-Tetradecen-1-ol (E)-11-Tetradecenyl acetate(E)-11-Tetradecenal (Z)-11-Tetradecen-1-ol (Z)-11-Tetradecenyl acetate(Z)-11-Tetradecenal (E)-12-Tetradecenyl acetate (Z)-12-Tetradecenylacetate (E,E)-2,4-Tetradecadienal (E,E)-3,5-Tetradecadienyl acetate(E,Z)-3,5-Tetradecadienyl acetate (Z,E)-3,5-Tetradecadienyl acetate(E,Z)-3,7-Tetradecadienyl acetate (E,Z)-3,8-Tetradecadienyl acetate(E,Z)-4,9-Tetradecadienyl acetate (E,Z)-4,9-Tetradecadienal(E,Z)-4,10-Tetradecadienyl acetate (E,E)-5,8-Tetradecadienal(Z,Z)-5,8-Tetradecadien-1-ol (Z,Z)-5,8-Tetradecadienyl acetate(Z,Z)-5,8-Tetradecadienal (E,E)-8,10-Tetradecadien-1-ol(E,E)-8,10-Tetradecadienyl acetate (E,E)-8,10-Tetradecadienal(E,Z)-8,10-Tetradecadienyl acetate (E,Z)-8,10-Tetradecadienal(Z,E)-8,10-Tetradecadien-1-ol (Z,E)-8,10-Tetradecadienyl acetate(Z,Z)-8,10-Tetradecadienal (E,E)-9,11-Tetradecadienyl acetate(E,Z)-9,11-Tetradecadienyl acetate (Z,E)-9,11-Tetradecadien-1-ol(Z,E)-9,11-Tetradecadienyl acetate (Z,E)-9,11-Tetradecadienal(Z,Z)-9,11-Tetradecadien-1-ol (Z,Z)-9,11-Tetradecadienyl acetate(Z,Z)-9,11-Tetradecadienal (E,E)-9,12-Tetradecadienyl acetate(Z,E)-9,12-Tetradecadien-1-ol (Z,E)-9,12-Tetradecadienyl acetate(Z,E)-9,12-Tetradecadienal (Z,Z)-9,12-Tetradecadien-1-ol(Z,Z)-9,12-Tetradecadienyl acetate (E,E)-10,12-Tetradecadien-1-ol(E,E)-10,12-Tetradecadienyl acetate (E,E)-10,12-Tetradecadienal(E,Z)-10,12-Tetradecadienyl acetate (Z,E)-10,12-Tetradecadienyl acetate(Z,Z)-10,12-Tetradecadien-1-ol (Z,Z)-10,12-Tetradecadienyl acetate(E,Z,Z)-3,8,11-Tetradecatrienyl acetate (E)-8-Pentadecen-1-ol(E)-8-Pentadecenyl acetate (Z)-8-Pentadecen-1-ol (Z)-8-Pentadecenylacetate (Z)-9-Pentadecenyl acetate (E)-9-Pentadecenyl acetate(Z)-10-Pentadecenyl acetate (Z)-10-Pentadecenal (E)-12-Pentadecenylacetate (Z)-12-Pentadecenyl acetate (Z,Z)-6,9-Pentadecadien-1-ol(Z,Z)-6,9-Pentadecadienyl acetate (Z,Z)-6,9-Pentadecadienal(E,E)-8,10-Pentadecadienyl acetate (E,Z)-8,10-Pentadecadien-1-ol(E,Z)-8,10-Pentadecadienyl acetate (Z,E)-8,10-Pentadecadienyl acetate(Z,Z)-8,10-Pentadecadienyl acetate (E,Z)-9,11-Pentadecadienal(Z,Z)-9,11-Pentadecadienal (Z)-3-Hexadecenyl acetate(E)-5-Hexadecen-1-ol (E)-5-Hexadecenyl acetate (Z)-5-Hexadecen-1-ol(Z)-5-Hexadecenyl acetate (E)-6-Hexadecenyl acetate (E)-7-Hexadecen-1-ol(E)-7-Hexadecenyl acetate (E)-7-Hexadecenal (Z)-7-Hexadecen-1-ol(Z)-7-Hexadecenyl acetate (Z)-7-Hexadecenal (E)-8-Hexadecenyl acetate(E)-9-Hexadecen-1-ol (E)-9-Hexadecenyl acetate (E)-9-Hexadecenal(Z)-9-Hexadecen-1-ol (Z)-9-Hexadecenyl acetate (Z)-9-Hexadecenal(E)-10-Hexadecen-1-ol (E)-10-Hexadecenal (Z)-10-Hexadecenyl acetate(Z)-10-Hexadecenal (E)-11-Hexadecen-1-ol (E)-11-Hexadecenyl acetate(E)-11-Hexadecenal (Z)-11-Hexadecen-1-ol (Z)-11-Hexadecenyl acetate(Z)-11-Hexadecenal (Z)-12-Hexadecenyl acetate (Z)-12-Hexadecenal(E)-14-Hexadecenal (Z)-14-Hexadecenyl acetate(E,E)-1,3-Hexadecadien-1-ol (E,Z)-4,6-Hexadecadien-1-ol(E,Z)-4,6-Hexadecadienyl acetate (E,Z)-4,6-Hexadecadienal(E,Z)-6,11-Hexadecadienyl acetate (E,Z)-6,11-Hexadecadienal(Z,Z)-7,10-Hexadecadien-1-ol (Z,Z)-7,10-Hexadecadienyl acetate(Z,E)-7,11-Hexadecadien-1-ol (Z,E)-7,11-Hexadecadienyl acetate(Z,E)-7,11-Hexadecadienal (Z,Z)-7,11-Hexadecadien-1-ol(Z,Z)-7,11-Hexadecadienyl acetate (Z,Z)-7,11-Hexadecadienal(Z,Z)-8,10-Hexadecadienyl acetate (E,Z)-8,11-Hexadecadienal(E,E)-9,11-Hexadecadienal (E,Z)-9,11-Hexadecadienyl acetate(E,Z)-9,11-Hexadecadienal (Z,E)-9,11-Hexadecadienal(Z,Z)-9,11-Hexadecadienal (E,E)-10,12-Hexadecadien-1-ol(E,E)-10,12-Hexadecadienyl acetate (E,E)-10,12-Hexadecadienal(E,Z)-10,12-Hexadecadien-1-ol (E,Z)-10,12-Hexadecadienyl acetate(E,Z)-10,12-Hexadecadienal (Z,E)-10,12-Hexadecadienyl acetate(Z,E)-10,12-Hexadecadienal (Z,Z)-10,12-Hexadecadienal(E,E)-11,13-Hexadecadien-1-ol (E,E)-11,13-Hexadecadienyl acetate(E,E)-11,13-Hexadecadienal (E,Z)-11,13-Hexadecadien-1-ol(E,Z)-11,13-Hexadecadienyl acetate (E,Z)-11,13-Hexadecadienal(Z,E)-11,13-Hexadecadien-1-ol (Z,E)-11,13-Hexadecadienyl acetate(Z,E)-11,13-Hexadecadienal (Z,Z)-11,13-Hexadecadien-1-ol(Z,Z)-11,13-Hexadecadienyl acetate (Z,Z)-11,13-Hexadecadienal(E,E)-10,14-Hexadecadienal (Z,E)-11,14-Hexadecadienyl acetate(E,E,Z)-4,6,10-Hexadecatrien-1-ol (E,E,Z)-4,6,10-Hexadecatrienyl acetate(E,Z,Z)-4,6,10-Hexadecatrien-1-ol (E,Z,Z)-4,6,10-Hexadecatrienyl acetate(E,E,Z)-4,6,11-Hexadecatrienyl acetate (E,E,Z)-4,6,11-Hexadecatrienal(Z,Z,E)-7,11,13-Hexadecatrienal (E,E,E)-10,12,14-Hexadecatrienyl acetate(E,E,E)-10,12,14-Hexadecatrienal (E,E,Z)-10,12,14-Hexadecatrienylacetate (E,E,Z)-10,12,14-Hexadecatrienal(E,E,Z,Z)-4,6,11,13-Hexadecatetraenal (E)-2-Heptadecenal(Z)-2-Heptadecenal (E)-8-Heptadecen-1-ol (E)-8-Heptadecenyl acetate(Z)-8-Heptadecen-1-ol (Z)-9-Heptadecenal (E)-10-Heptadecenyl acetate(Z)-11-Heptadecen-1-ol (Z)-11-Heptadecenyl acetate(E,E)-4,8-Heptadecadienyl acetate (Z,Z)-8,10-Heptadecadien-1-ol(Z,Z)-8,11-Heptadecadienyl acetate (E)-2-Octadecenyl acetate(E)-2-Octadecenal (Z)-2-Octadecenyl acetate (Z)-2-Octadecenal(E)-9-Octadecen-1-ol (E)-9-Octadecenyl acetate (E)-9-Octadecenal(Z)-9-Octadecen-1-ol (Z)-9-Octadecenyl acetate (Z)-9-Octadecenal(E)-11-Octadecen-1-ol (E)-11-Octadecenal (Z)-11-Octadecen-1-ol(Z)-11-Octadecenyl acetate (Z)-11-Octadecenal (E)-13-Octadecenyl acetate(E)-13-Octadecenal (Z)-13-Octadecen-1-ol (Z)-13-Octadecenyl acetate(Z)-13-Octadecenal (E)-14-Octadecenal (E,Z)-2,13-Octadecadien-1-ol(E,Z)-2,13-Octadecadienyl acetate (E,Z)-2,13-Octadecadienal(Z,E)-2,13-Octadecadienyl acetate (Z,Z)-2,13-Octadecadien-1-ol(Z,Z)-2,13-Octadecadienyl acetate (E,E)-3,13-Octadecadienyl acetate(E,Z)-3,13-Octadecadienyl acetate (E,Z)-3,13-Octadecadienal(Z,E)-3,13-Octadecadienyl acetate (Z,Z)-3,13-Octadecadienyl acetate(Z,Z)-3,13-Octadecadienal (E,E)-5,9-Octadecadien-1-ol(E,E)-5,9-Octadecadienyl acetate (E,E)-9,12-Octadecadien-1-ol(Z,Z)-9,12-Octadecadienyl acetate (Z,Z)-9,12-Octadecadienal(Z,Z)-11,13-Octadecadienal (E,E)-11,14-Octadecadienal(Z,Z)-13,15-Octadecadienal (Z,Z,Z)-3,6,9-Octadecatrienyl acetate(E,E,E)-9,12,15-Octadecatrien-1-ol (Z,Z,Z)-9,12,15-Octadecatrienylacetate (Z,Z,Z)-9,12,15-Octadecatrienal

In some aspects, the pheromones synthesized as taught in this disclosureinclude at least one pheromone listed in Table 2a to modulate thebehavior of an insect listed in Table 2a. In other aspects, non-limitingexamples of insect pheromones which can be synthesized using therecombinant microorganisms and methods disclosed herein includealcohols, aldehydes, and acetates listed in Table 2a. However, themicroorganisms described herein are not limited to the synthesis ofC₆-C₂₀ pheromones listed in Table 1 and Table 2a. Rather, the disclosedmicroorganisms can also be utilized in the synthesis of various C₆-C₂₄mono- or poly-unsaturated fatty alcohols, aldehydes, and acetates,including fragrances, flavors, and polymer intermediates.

TABLE 2a Exemplary pheromones that can be synthesized according tomethods described in the present disclosure. Example of Biological NameStructure importance (Z)-3-hexen-1-ol

See, Sugimoto et al. (2014) (Z)-3-nonen-1-ol

West Indian Fruity Fly male sex pheromone (Z)-5-decen-1-ol

(Z)-5-decenyl acetate

Agrotis segetum sex pheromone component (E)-5-decen-1-ol

Anarsia lineatella sex pheromone component (E)-5-decenyl acetate

Anarsia lineatella sex pheromone component (Z)-7-dodecen-1-ol

(Z)-7-dodecenyl acetate

Pseudoplusia includens sex pheromone Agrotis segetum sex pheromonecomponent (E)-8-dodecen-1-ol

Citrus Fruit Moth sex pheromone (E)-8-dodecenyl acetate

Grapholitha molesta, Ecdytolopha aurantiana sex pheromone component(Z)-8-dodecen-1-ol

Grapholitha molesta, Ecdytolopha aurantiana sex pheromone component(Z)-8-dodecenyl acetate

Grapholitha molesta sex pheromone component (Z)-9-dodecen-1-ol

(Z)-9-dodecenyl acetate

Eupoecilia ambiguella sex pheromone (E,E)-8,10-dodecadien- 1-ol

Cydia pomonella (7E,9Z)-dodecadienyl acetate

Lobesia botrana (Z)-9-tetradecen-1-ol

(Z)-9-tetradecenyl acetate

Pandemis pyrusana, Naranga aenescens, Agrotis segetum sex pheromonecomponent (Z)-11-tetradecen-1-ol

(Z)-11-tetradecenyl acetate

Pandemis pyrusana, Choristoneura roseceana sex pheromone component(E)-11-tetradecen-1-ol

(E)-11-tetradecenyl acetate

Choristoneura roseceana, Crocidolomia pavonana sex pheromone component(Z)-7-hexadecen-1-ol

(Z)-7-hexadecenal

Diatraea considerata sex pheromone component (Z)-9-hexadecen-1-ol

(Z)-9-hexadecenal

Helicoverpa zea, Helicoverpa armigera, Heliothis virescens sex pheromonecomponent (Z)-9-hexadecenyl acetate

Naranga aenescens sex pheromone component (Z)-11-hexadecen-1-ol

(Z)-11-hexadecenal

Platyptila carduidactyla, Heliothis virescens sex pheromone Helicoverpazea, Helicoverpa armigera, Plutella xylostella, Diatraea considerate,Diatraea grandiosella, Diatraea saccharalis, Acrolepiopsis assectellasex pheromone component (Z)-11-hexadecenyl acetate

Discestra trifolii sex pheromone Heliothis virescens, Plutellaxylostella, Acrolepiopsis assectella, Crocidolomia pavonana, Narangaaenescens sex pheromone component (Z,Z)-11,13- hexadecadienal

Amyelosis transitella (Z,Z)-11,13- hexadecadien-1-ol

Amyelosis transitella (11Z,13E)- hexadecadien-1-ol

Amyelosis transitella (9Z,11E)- hexadecadienal

(Z)-13-octadecen-1-ol

(Z)-13-octadecenal

Diatraea considerata, Diatraea grandiosella sex pheromone component(Z,Z,Z,Z,Z)-3,6,9,12,15- tricosapentaene

Amyelosis transitella

Most pheromones comprise a hydrocarbon skeleton with the terminalhydrogen substituted by a functional group (Ryan M F (2002). InsectChemoreception. Fundamental and Applied. Kluwer Academic Publishers).Table 2b shows some common functional groups, along with their formulas,prefixes and suffixes. The presence of one or more double bonds,generated by the loss of hydrogens from adjacent carbons, determines thedegree of unsaturation of the molecule and alters the designation of ahydrocarbon from -ane (no multiple bonds) to -ene. The presence of twoand three double bonds is indicated by ending the name with -diene and-triene, respectively. The position of each double bond is representedby a numeral corresponding to that of the carbon from which it begins,with each carbon numbered from that attached to the functional group.The carbon to which the functional group is attached is designated -1-.Pheromones may have, but are not limited to, hydrocarbon chain lengthsnumbering 10 (deca-), 12 (dodeca-), 14 (tetradeca-), 16 (hexadeca-) , or18 (octadeca-) carbons long. The presence of a double bond has anothereffect. It precludes rotation of the molecule by fixing it in one of twopossible configurations, each representing geometric isomers that aredifferent molecules. These are designated either E (from the German wordEntgegen, opposite) or Z (Zusammen, together), when the carbon chainsare connected on the opposite (trans) or same (cis) side, respectively,of the double bond.

TABLE 2b Prefixes and suffixes for common functional groups Functionalgroup Formula Prefix Suffix Alcohol —OH Hydroxy- -ol Aldehyde —CH═OFormyl- -al Amine —NH₂ Amino- -amine Carboxylic acid —COOH Carboxy- -oicacid Ester —COOR R-oxycarbonyl- -R-oate Ketone >C═O Oxo- -one|From Howse, P E, Stevens, I D R and Jones, O T (1998). Insect pheromonesand their use in pest management. London: Chapman and Hall.

Pheromones described herein can be referred to using IUPAC nomenclatureor various abbreviations or variations known to one skilled in the art.For example, (11Z)-hexadecen-1-al, can also be written asZ-11-hexadecen-1-al, Z-11-hexadecenal, or Z-x-y:Ald, wherein xrepresents the position of the double bond and y represents the numberof carbons in the hydrocarbon skeleton. Abbreviations used herein andknown to those skilled in the art to identify functional groups on thehydrocarbon skeleton include “Ald,” indicating an aldehyde, “OH,”indicating an alcohol, and “Ac,” indicating an acetyl. Also, the numberof carbons in the chain can be indicated using numerals rather thanusing the written name. Thus, as used herein, an unsaturated carbonchain comprised of sixteen carbons can be written as hexadecene or 16.

Similar abbreviation and derivations are used herein to describepheromone precursors. For example, the fatty acyl-CoA precursors of(11Z)-hexadecen-1-al can be identified as (11Z)-hexadecenyl-CoA orZ-11-16:Acyl-CoA.

The present disclosure relates to the synthesis of mono- orpoly-unsaturated C₆-C₂₄ fatty alcohols, aldehydes, and acetates using arecombinant microorganism comprised of one or more heterologous enzymes,which catalyze substrate to product conversions for one or more steps inthe synthesis process.

Desaturase

The present disclosure describes enzymes that desaturate fatty acylsubstrates to corresponding unsaturated fatty acyl substrates.

In some embodiments, a desaturase is used to catalyze the conversion ofa fatty acyl-CoA or acyl-ACP to a corresponding unsaturated fattyacyl-CoA or acyl-ACP. A desaturase is an enzyme that catalyzes theformation of a carbon-carbon double bond in a saturated fatty acid orfatty acid derivative, e.g., fatty acyl-CoA or fatty acyl-ACP(collectively referred to herein as “fatty acyl”), by removing at leasttwo hydrogen atoms to produce a corresponding unsaturated fattyacid/acyl. Desaturases are classified with respect to the ability of theenzyme to selectively catalyze double bond formation at a subterminalcarbon relative to the methyl end of the fatty acid/acyl or asubterminal carbon relative to the carbonyl end of the fatty acid/acyl.Omega (ω) desaturases catalyze the formation of a carbon-carbon doublebond at a fixed subterminal carbon relative to the methyl end of a fattyacid/acyl. For example, an ω³ desaturase catalyzes the formation of adouble bond between the third and fourth carbon relative the methyl endof a fatty acid/acyl. Delta (4) desaturases catalyze the formation of acarbon-carbon double bond at a specific position relative to thecarboxyl group of a fatty acid or the carbonyl group of a fatty acylCoA. For example, a Δ⁹ desaturase catalyzes the formation of a doublebond between the C₉ and C₁₀ carbons with respect to the carboxyl end ofthe fatty acid or the carbonyl group of a fatty acyl CoA.

As used herein, a desaturase can be described with reference to thelocation in which the desaturase catalyzes the formation of a doublebond and the resultant geometric configuration (i.e., E/Z) of theunsaturated hydrocarbon. Accordingly, as used herein, a Z9 desaturaserefers to a Δ desaturase that catalyzes the formation of a double bondbetween the C₉ and C₁₀ carbons with respect to the carbonyl end of afatty acid/acyl, thereby orienting two hydrocarbons on opposing sides ofthe carbon-carbon double bonds in the cis or Z configuration. Similarly,as used herein, a Z11 desaturase refers to a Δ desaturase that catalyzesthe formation of a double bond between the C₁₁ and C₁₂ carbons withrespect to the carbonyl end of a fatty acid/acyl.

Desaturases have a conserved structural motif. This sequence motif oftransmembrane desaturases is characterized by [HX3-4HX7-41(3non-His)HX2-3(1 nonHis)HHX61-189(40 non-His)HX2-3(1 non-His)HH]. Thesequence motif of soluble desaturases is characterized by twooccurrences of [D/EEXXH].

In some embodiments, the desaturase is a fatty acyl-CoA desaturase thatcatalyzes the formation of a double bond in a fatty acyl-CoA. In somesuch embodiments, the fatty acyl-CoA desaturase described herein iscapable of utilizing a fatty acyl-CoA as a substrate that has a chainlength of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, or 24 carbon atoms. Thus, the desaturase used in the recombinantmicroorganism can be selected based on the chain length of thesubstrate.

In some embodiments, the fatty acyl desaturase described herein iscapable of catalyzing the formation of a double bond at a desired carbonrelative to the terminal CoA on the unsaturated fatty acyl-CoA. Thus, insome embodiments, a desaturase can be selected for use in therecombinant microorganism which catalyzes double bond insertion at the3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 position with respect to thecarbonyl group on a fatty acyl-CoA.

In some embodiments, the fatty acyl desaturase described herein iscapable of catalyzing the formation of a double bond in a saturatedfatty acyl-CoA such that the resultant unsaturated fatty acyl-CoA has acis or trans (i.e., Z or E) geometric configuration.

In some embodiments, the desaturase is a fatty acyl-ACP desaturase thatcatalyzes the formation of a double bond in a fatty acyl-ACP. In someembodiments, the fatty acyl-ACP desaturase described herein is capableof utilizing a fatty acyl-CoA as a substrate that has a chain length of6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or24 carbon atoms. Thus, the desaturase used in the recombinantmicroorganism can be selected based on the chain length of thesubstrate.

In some embodiments, the fatty acyl-ACP desaturase described herein iscapable of catalyzing the formation of a double bond at a desired carbonrelative to the terminal carbonyl on the unsaturated fatty acyl-ACP.Thus, in some embodiments, a desaturase can be selected for use in therecombinant microorganism which catalyzes double bond insertion at the3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 position with respect to thecarbonyl group on a fatty acyl-ACP.

In some embodiments, the fatty acyl desaturase described herein iscapable of catalyzing the formation of a double bond in a saturatedfatty acyl-CoA such that the resultant unsaturated fatty acyl-ACP has acis or trans (i.e., Z or E) geometric configuration.

In one embodiment, the fatty acyl desaturase is a Z11 desaturase. Insome embodiments, a nucleic acid sequence encoding a Z11 desaturase fromorganisms of the species Agrotis segetum, Amyelois transitella,Argyrotaenia velutiana, Choristoneura rosaceana, Lampronia capitella,Trichoplusia ni, Helicoverpa zea, or Thalassiosira pseudonana is codonoptimized. In some embodiments, the Z11 desaturase comprises a sequenceselected from SEQ ID NOs: 9, 18, 24 and 26 from Trichoplusia ni. Inother embodiments, the Z11 desaturase comprises a sequence selected fromSEQ ID NOs: 10 and 16 from Agrotis segetum. In some embodiments, the Z11desaturase comprises a sequence selected from SEQ ID NOs: 11 and 23 fromThalassiosira pseudonana. In certain embodiments, the Z11 desaturasecomprises a sequence selected from SEQ ID NOs: 12, 17 and 30 fromAmyelois transitella. In further embodiments, the Z11 desaturasecomprises a sequence selected from SEQ ID NOs: 13, 19, 25, 27 and 31from Helicoverpa zea. In some embodiments, the Z11 desaturase comprisesa chimeric polypeptide. In some embodiments, a complete or partial Z11desaturase is fused to another polypeptide. In certain embodiments, theN-terminal native leader sequence of a Z11 desaturase is replaced by anoleosin leader sequence from another species. In certain embodiments,the Z11 desaturase comprises a sequence selected from SEQ ID NOs: 15, 28and 29.

In one embodiment, the fatty acyl desaturase is a Z9 desaturase. In someembodiments, a nucleic acid sequence encoding a Z9 desaturase is codonoptimized. In some embodiments, the Z9 desaturase comprises a sequenceset forth in SEQ ID NO: 20 from Ostrinia furnacalis. In otherembodiments, the Z9 desaturase comprises a sequence set forth in SEQ IDNO: 21 from Lampronia capitella. In some embodiments, the Z9 desaturasecomprises a sequence set forth in SEQ ID NO: 22 from Helicoverpa zea.

Fatty Acyl Reductase

The present disclosure describes enzymes that reduce fatty acylsubstrates to corresponding fatty alcohols or aldehydes.

In some embodiments, a fatty alcohol forming fatty acyl-reductase isused to catalyze the conversion of a fatty acyl-CoA to a correspondingfatty alcohol. In some embodiments, a fatty aldehyde forming fattyacyl-reductase is used to catalyze the conversion of a fatty acyl-ACP toa corresponding fatty aldehyde. A fatty acyl reductase is an enzyme thatcatalyzes the reduction of a fatty acyl-CoA to a corresponding fattyalcohol or the reduction of a fatty acyl-ACP to a corresponding fattyaldehyde. A fatty acyl-CoA and fatty acyl-ACP has a structure ofR—(CO)—S—R₁, wherein R is a C₆ to C₂₄ saturated, unsaturated, linear,branched or cyclic hydrocarbon, and R₁ represents CoA or ACP. In aparticular embodiment, R is a C₆ to C₂₄ saturated or unsaturated linearhydrocarbon. “CoA” is a non-protein acyl carrier group involved in thesynthesis and oxidation of fatty acids. “ACP” is an acyl carrierprotein, i.e., a polypeptide or protein subunit, of fatty acid synthaseused in the synthesis of fatty acids.

Thus, in some embodiments, the disclosure provides for a fatty alcoholforming fatty acyl-reductase which catalyzes the reduction of a fattyacyl-CoA to the corresponding fatty alcohol. For example, R—(CO)—S-CoAis converted to R—CH₂OH and CoA-SH when two molecules of NAD(P)H areoxidized to NAD(P)⁺. Accordingly, in some such embodiments, arecombinant microorganism described herein can include a heterologousfatty alcohol forming fatty acyl-reductase, which catalyzes thereduction a fatty acyl-CoA to the corresponding fatty alcohol. In anexemplary embodiment, a recombinant microorganism disclosed hereinincludes at least one exogenous nucleic acid molecule encoding a fattyalcohol forming fatty-acyl reductase which catalyzes the conversion of amono- or poly-unsaturated C₆-C₂₄ fatty acyl-CoA into the correspondingmono- or poly-unsaturated C₆-C₂₄ fatty alcohol.

In other embodiments, the disclosure provides for a fatty aldehydeforming fatty acyl-reductase which catalyzes the reduction of a fattyacyl-ACP to the corresponding fatty aldehyde. For example, R—(CO)—S-ACPis converted to R—(CO)—H and ACP-SH when one molecule of NAD(P)H isoxidized to NAD(P)⁺. In some such embodiments, a recombinantmicroorganism described herein can include a heterologous fatty aldehydeforming fatty acyl-reductase, which catalyzes the reduction a fattyacyl-ACP to the corresponding fatty aldehyde. In an exemplaryembodiment, a recombinant microorganism disclosed herein includes atleast one exogenous nucleic acid molecule encoding a fatty aldehydeforming fatty-acyl reductase which catalyzes the conversion of a mono-or poly-unsaturated C₆-C₂₄ fatty acyl-ACP into the corresponding mono-or poly-unsaturated C₆-C₂₄ fatty aldehyde.

In some embodiments, a nucleic acid sequence encoding a fatty-acylreductase from organisms of the species Agrotis segetum, Spodopteralittoralis, or Helicoverpa amigera is codon optimized. In someembodiments, the fatty acyl reductase comprises a sequence set forth inSEQ ID NO: 1 from Agrotis segetum. In other embodiments, the fatty acylreductase comprises a sequence set forth in SEQ ID NO: 2 from Spodopteralittoralis. In some embodiments, the fatty acyl reductase comprises asequence selected from SEQ ID NOs: 3 and 32 from Helicoverpa armigera.

Acyl-ACP Synthetase

The present disclosure describes enzymes that ligate a fatty acid to thecorresponding fatty acyl-ACP.

In some embodiments, an acyl-ACP synthetase is used to catalyze theconversion of a fatty acid to a corresponding fatty acyl-ACP. Anacyl-ACP synthetase is an enzyme capable of ligating a fatty acid to ACPto produce a fatty acid acyl-ACP. In some embodiments, an acyl-ACPsynthetase can be used to catalyze the conversion of a fatty acid to acorresponding fatty acyl-ACP. In some embodiments, the acyl-ACPsynthetase is a synthetase capable of utilizing a fatty acid as asubstrate that has a chain length of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, or 24 carbon atoms. In one suchembodiment, a recombinant microorganism described herein can include aheterologous acyl-ACP synthetase, which catalyzes the conversion of afatty acid to a corresponding fatty acyl-ACP. In an exemplaryembodiment, a recombinant microorganism disclosed herein includes atleast one exogenous nucleic acid molecule which encodes an acyl-ACPsynthetase that catalyzes the conversion of a saturated C₆-C₂₄ fattyacid to a corresponding saturated C₆-C₂₄ fatty acyl-ACP.

Fatty Acid Synthase Complex

The present disclosure describes enzymes that catalyze the elongation ofa carbon chain in fatty acid.

In some embodiments, a fatty acid synthase complex is used to catalyzeinitiation and elongation of a carbon chain in a fatty acid. A “fattyacid synthase complex” refers to a group of enzymes that catalyzes theinitiation and elongation of a carbon chain on a fatty acid. The ACPalong with the enzymes in the fatty acid synthase (FAS) pathway controlthe length, degree of saturation, and branching of the fatty acidsproduced. The steps in this pathway are catalyzed by enzymes of thefatty acid biosynthesis (fab) and acetyl-CoA carboxylase (acc) genefamilies. Depending upon the desired product, one or more of these genescan be attenuated, expressed or over-expressed. In exemplaryembodiments, one or more of these genes is over-expressed.

There are two principal classes of fatty acid synthases. Type I (FAS I)systems utilize a single large, multifunctional polypeptide and arecommon to both mammals and fungi (although the structural arrangement offungal and mammalian synthases differ). The Type I FAS system is alsofound in the CMN group of bacteria (corynebacteria, mycobacteria, andnocardia). The Type II FAS (FAS II) is characterized by the use ofdiscrete, monofunctional enzymes for fatty acid synthesis, and is foundin archaea and bacteria.

The mechanism of FAS I and FAS II elongation and reduction is thesubstantially similar, as the domains of the FAS I multienzymepolypeptides and FAS II enzymes are largely conserved.

Fatty acids are synthesized by a series of decarboxylative Claisencondensation reactions from acetyl-CoA and malonyl-CoA. The steps inthis pathway are catalyzed by enzymes of the fatty acid biosynthesis(fab) and acetyl-CoA carboxylase (acc) gene families. For a descriptionof this pathway, see, e.g., Heath et al., Prog. Lipid Res. 40:467, 2001,which is herein incorporated by reference in its entirety. Without beinglimited by theory, in bacteria, acetyl-CoA is carboxylated by acetyl-CoAcarboxylase (Acc, a multi-subunit enzyme encoded by four separate genes,accABCD), to form malonyl-CoA. In yeast, acetyl-CoA is carboxylated bythe yeast equivalents of the acetyl-CoA carboxylase, encoded by ACC1 andACC2. In bacteria, the malonate group is transferred to ACP bymalonyl-CoA:ACP transacylase (FabD) to form malonyl-ACP. In yeast, amalonyl-palmityl tranferase domain adds malonyl from malonyl-CoA to theACP domain of the FAS complex. A condensation reaction then occurs,where malonyl-ACP merges with acyl-CoA, resulting in β-ketoacyl-ACP. Inthis manner, the hydrocarbon substrate is elongated by 2 carbons.

Following elongation, the β-keto group is reduced to the fully saturatedcarbon chain by the sequential action of a keto-reductase (KR),dehydratase (DH), and enol reductase (ER). The elongated fatty acidchain is carried between these active sites while attached covalently tothe phosphopantetheine prosthetic group of ACP. First, theβ-ketoacyl-ACP is reduced by NADPH to form β-hydroxyacyl-ACP. Inbacteria, this step is catalyzed by β-ketoacyl-ACP reductase (FabG). Theequivalent yeast reaction is catalyzed by the ketoreductase (KR) domainof FAS. β-hydroxyacyl-ACP is then dehydrated to form trans-2-enoyl-ACP,which is catalyzed by either β-hydroxyacyl-ACP dehydratase/isomerase(FabA) or β-hydroxyacyl-ACP dehydratase (FabZ) in bacteria or thedehydratase (DH) domain of FAS in yeast. NADPH-dependenttrans-2-enoyl-ACP reductase I, II, or III (Fabl, FabK, and FabL,respectively) in bacteria and the enol reductase (ER) domain of FAS inyeast reduces trans-2-enoyl-ACP to form acyl-ACP. Subsequent cycles arestarted by the condensation of malonyl-ACP with acyl-ACP byβ-ketoacyl-ACP synthase I or β-ketoacyl-ACP synthase II (FabB and FabF,respectively, in bacteria or the beta-ketoacyl synthase (KS) domain inyeast).

In some embodiments, a fatty acid synthase complex can be used tocatalyze elongation of a fatty acyl-ACP to a corresponding fattyacyl-ACP with a two carbon elongation relative to the substrate.

Dehydrogenase

The present disclosure describes enzymes that catalyze the conversion ofa fatty aldehyde to a fatty alcohol. In some embodiments, an alcoholdehydrogenase (ADH, Table 3) is used to catalyze the conversion of afatty aldehyde to a fatty alcohol. A number of ADHs identified fromalkanotrophic organisms, Pseudomonas fluorescens NRRL B-1244 (Hou et al.1983), Pseudomonas butanovora ATCC 43655 (Vangnai and Arp 2001), andAcinetobacter sp. strain M-1 (Tani et al. 2000), have shown to be activeon short to medium-chain alkyl alcohols (C₂ to C₁₄). Additionally,commercially available ADHs from Sigma, Horse liver ADH and Baker'syeast ADH have detectable activity for substrates with length C₁₀ andgreater. The reported activities for the longer fatty alcohols may beimpacted by the difficulties in solubilizing the substrates. For theyeast ADH from Sigma, little to no activity is observed for C₁₂ to C₁₄aldehydes by (Tani et al. 2000), however, activity for C₁₂ and C₁₆hydroxy-w-fatty acids has been observed (Lu et al. 2010). Recently, twoADHs were characterized from Geobacillus thermodenitrificans NG80-2, anorganism that degrades C₁₅ to C₃₆ alkanes using the LadA hydroxylase.Activity was detected from methanol to 1-triacontanol (C₃₀) for bothADHs, with 1-octanol being the preferred substrate for ADH2 and ethanolfor ADH1 (Liu et al. 2009).

The use of ADHs in whole-cell bioconversions has been mostly focused onthe production of chiral alcohols from ketones (Ernst et al. 2005)(Schroer et al. 2007). Using the ADH from Lactobacillus brevis andcoupled cofactor regeneration with isopropanol, Schroer et al. reportedthe production of 797 g of (R)-methyl-3 hydroxybutanoate from methylacetoacetate, with a space time yield of 29 g/L/h (Schroer et al. 2007).Examples of aliphatic alcohol oxidation in whole-cell transformationshave been reported with commercially obtained S. cerevisiae for theconversion of hexanol to hexanal (Presecki et al. 2012) and 2-heptanolto 2-heptanone (Cappaert and Larroche 2004).

TABLE 3 Exemplary alcohol dehydrogenase enzymes. Accession Organism GeneName No. Bactrocera oleae (Olive fruit fly) (Dacus ADH Q9NAR7 oleae)Cupriavidus necator (Alcaligenes adh P14940 eutrophus) (Ralstoniaeutropha) Drosophila adiastola (Fruit fly) (Idiomyia Adh Q00669adiastola) Drosophila affinidisjuncta (Fruit fly) Adh P21518 (Idiomyiaaffinidisjuncta) Drosophila ambigua (Fruit fly) Adh P25139 Drosophilaborealis (Fruit fly) Adh P48584 Drosophila differens (Fruit fly) AdhP22245 Drosophila equinoxialis (Fruit fly) Adh Q9NG42 Drosophilaflavomontana (Fruit fly) Adh P48585 Drosophila guanche (Fruit fly) AdhQ09009 Drosophila hawaiiensis (Fruit fly) Adh P51549 Drosophilaheteroneura (Fruit fly) Adh P21898 Drosophila immigrans (Fruit fly) AdhQ07588 Drosophila insularis (Fruit fly) Adh Q9NG40 Drosophilalebanonensis (Fruit fly) Adh P10807 (Scaptodrosophila lebanonensis)Drosophila mauritiana (Fruit fly) Adh P07162 Drosophila madeirensis(Fruit fly) Adh Q09010 Drosophila mimica (Fruit fly) (Idiomyia AdhQ00671 mimica) Drosophila nigra (Fruit fly) (Idiomyia Adh Q00672 nigra)Drosophila orena (Fruit fly) Adh P07159 Drosophila pseudoobscurabogotana Adh P84328 (Fruit fly) Drosophila picticornis (Fruit fly) AdhP23361 (Idiomyia picticornis) Drosophila planitibia (Fruit fly) AdhP23277 Drosophila paulistorum (Fruit fly) Adh Q9U8S9 Drosophilasilvestris (Fruit fly) Adh P23278 Drosophila subobscura (Fruit fly) AdhQ03384 Drosophila teissieri (Fruit fly) Adh P28484 Drosophila tsacasi(Fruit fly) Adh P51550 Fragaria ananassa (Strawberry) ADH P17648 Malusdomestica (Apple) (Pyrus malus) ADH P48977 Scaptomyza albovittata (Fruitfly) Adh P25988 Scaptomyza crassifemur (Fruit fly) Adh Q00670(Drosophila crassifemur) Sulfolobus sp. (strain RC3) adh P50381Zaprionus tuberculatus (Vinegar fly) Adh P51552 Geobacillusstearothermophilus (Bacillus adh P42327 stearothermophilus) Drosophilamayaguana (Fruit fly) Adh, Adh2 P25721 Drosophila melanogaster (Fruitfly) Adh, CG3481 P00334 Drosophila pseudoobscura pseudoobscura Adh,GA17214 Q6LCE4 (Fruit fly) Drosophila simulans (Fruit fly) Adh, GD23968Q24641 Drosophila yakuba (Fruit fly) Adh, GE19037 P26719 Drosophilaananassae (Fruit fly) Adh, GF14888 Q50L96 Drosophila erecta (Fruit fly)Adh, GG25120 P28483 Drosophila grimshawi (Fruit fly) Adh, GH13025 P51551(Idiomyia grimshawi) Drosophila willistoni (Fruit fly) Adh, GK18290Q05114 Drosophila persimilis (Fruit fly) Adh, GL25993 P37473 Drosophilasechellia (Fruit fly) Adh, GM15656 Q9GN94 Cupriavidus necator (strainATCC 17699/ adh, H16_A0757 Q0KDL6 H16/DSM 428/Stanier 337) (Ralstoniaeutropha) Mycobacterium tuberculosis (strain CDC adh, MT1581 P9WQC21551/Oshkosh) Staphylococcus aureus (strain MW2) adh, MW0568 Q8NXU1Mycobacterium tuberculosis (strain ATCC adh, Rv1530 P9WQC3 25618/H37Rv)Staphylococcus aureus (strain N315) adh, SA0562 Q7A742 Staphylococcusaureus (strain bovine adh, SAB0557 Q2YSX0 RF122/ET3-1) Sulfolobusacidocaldarius (strain ATCC adh, Saci_2057 Q4J781 33909/DSM 639/JCM8929/NBRC 15157/NCIMB 11770) Staphylococcus aureus (strain COL) adh,SACOL0660 Q5HI63 Staphylococcus aureus (strain NCTC adh, Q2G0G1 8325)SAOUHSC_00608 Staphylococcus aureus (strain MRSA252) adh, SAR0613 Q6GJ63Staphylococcus aureus (strain M55A476) adh, SA50573 Q6GBM4Staphylococcus aureus (strain USA300) adh, Q2FJ31 SAUSA300_0594Staphylococcus aureus (strain Mu50/ adh, SAV0605 Q99W07 ATCC 700699)Staphylococcus epidermidis (strain ATCC adh, SE_0375 Q8CQ56 12228)Staphylococcus epidermidis (strain ATCC adh, SERP0257 Q5HRD635984/RP62A) Sulfolobus solfataricus (strain ATCC adh, SSO2536 P3946235092/DSM 1617/JCM 11322/P2) Sulfolobus tokodaii (strain DSM 16993/ adh,STK_25770 Q96XE0 JCM 10545/NBRC 100140/7) Anas platyrhynchos (Domesticduck) ADH1 P30350 (Anas boschas) Apteryx australis (Brown kiwi) ADH1P49645 Ceratitis capitata (Mediterranean fruit fly) ADH1 P48814(Tephritis capitata) Ceratitis cosyra (Mango fruit fly) (Trypeta ADH1Q70UN9 cosyra) Gallus gallus (Chicken) ADH1 P23991 Columba livia(Domestic pigeon) ADH1 P86883 Coturnix coturnix japonica (Japanese ADH1P19631 quail) (Coturnix japonica) Drosophila hydei (Fruit fly) Adh1P23236 Drosophila montana (Fruit fly) Adh1 P48586 Drosophila mettleri(Fruit fly) Adh1 P22246 Drosophila mulleri (Fruit fly) Adh1 P07161Drosophila navojoa (Fruit fly) Adh1 P12854 Geomys attwateri (Attwater'spocket ADH1 Q9Z2M2 gopher) (Geomys bursarius attwateri) Geomys bursarius(Plains pocket gopher) ADH1 Q64413 Geomys knoxjonesi (Knox Jones'spocket ADH1 Q64415 gopher) Hordeum vulgare (Barley) ADH1 P05336Kluyveromyces marxianus (Yeast) ADH1 Q07288 (Candida kefyr) Zea mays(Maize) ADH1 P00333 Mesocricetus auratus (Golden hamster) ADH1 P86885Pennisetum americanum (Pearl millet) ADH1 P14219 (Pennisetum glaucum)Petunia hybrida (Petunia) ADH1 P25141 Oryctolagus cuniculus (Rabbit)ADH1 Q03505 Solanum tuberosum (Potato) ADH1 P14673 Struthio camelus(Ostrich) ADH1 P80338 Trifolium repens (Creeping white clover) ADH1P13603 Zea luxurians (Guatemalan teosinte) ADH1 Q07264 (Euchlaenaluxurians) Saccharomyces cerevisiae (strain ATCC ADH1, ADC1, P00330204508/S288c) (Baker's yeast) YOL086C, O0947 Arabidopsis thaliana(Mouse-ear cress) ADH1, ADH, P06525 At1g77120, F22K20.19Schizosaccharomyces pombe (strain 972/ adh1, adh, P00332 ATCC 24843)(Fission yeast) SPCC13B11.01 Drosophila lacicola (Fruit fly) Adh1, Adh-1Q27404 Mus musculus (Mouse) Adh1, Adh-1 P00329 Peromyscus maniculatus(North American ADH1, ADH-1 P41680 deer mouse) Rattus norvegicus (Rat)Adh1, Adh-1 P06757 Drosophila virilis (Fruit fly) Adh1, Adh-1, B4M8Y0GJ18208 Scheffersomyces stipitis (strain ATCC ADH1, ADH2, O0009758785/CBS 6054/NBRC 10063/NRRL PICST_68558 Y-11545) (Yeast) (Pichiastipitis) Aspergillus flavus (strain ATCC 200026/ adh1, P41747 FGSCA1120/NRRL 3357/JCM 12722/ AFLA_048690 SRRC 167) Neurospora crassa(strain ATCC 24698/ adh-1, Q9P6C8 74-OR23-1A/CBS 708.71/DSM 1257/B17C10.210, FGSC 987) NCU01754 Candida albicans (Yeast) ADH1, CAD P43067Oryza sativa subsp. japonica (Rice) ADH1, DUPR11.3, Q2R8Z5 Os11g0210300,LOC_Os11g10480, OsJ_032001 Drosophila mojavensis (Fruit fly) Adh1,GI17644 P09370 Kluyveromyces lactis (strain ATCC 8585/ ADH1, P20369 CBS2359/DSM 70799/NBRC 1267/ KLLA0F21010g NRRL Y-1140/WM37) (Yeast)(Candida sphaerica) Oryza sativa subsp. indica (Rice) ADH1, Q75ZX4OsI_034290 Pongo abelii (Sumatran orangutan) ADH1A Q5RBP7 (Pongopygmaeus abelii) Homo sapiens (Human) ADH1A, ADH1 P07327 Macaca mulatta(Rhesus macaque) ADH1A, ADH1 P28469 Pan troglodytes (Chimpanzee) ADH1BQ5R1W2 Papio hamadryas (Hamadryas baboon) ADH1B P14139 Homo sapiens(Human) ADH1B, ADH2 P00325 Homo sapiens (Human) ADH1C, ADH3 P00326 Papiohamadryas (Hamadryas baboon) ADH1C, ADH3 O97959 Ceratitis capitata(Mediterranean fruit ADH2 P48815 fly) (Tephritis capitata) Ceratitiscosyra (Mango fruit fly) ADH2 Q70UP5 (Trypeta cosyra) Ceratitis rosa(Natal fruit fly) ADH2 Q70UP6 (Pterandrus rosa) Drosophila arizonae(Fruit fly) Adh2 P27581 Drosophila buzzatii (Fruit fly) Adh2 P25720Drosophila hydei (Fruit fly) Adh2 P23237 Drosophila montana (Fruit fly)Adh2 P48587 Drosophila mulleri (Fruit fly) Adh2 P07160 Drosophilawheeleri (Fruit fly) Adh2 P24267 Entamoeba histolytica ADH2 Q24803Hordeum vulgare (Barley) ADH2 P10847 Kluyveromyces marxianus (Yeast)ADH2 Q9P4C2 (Candida kefyr) Zea mays (Maize) ADH2 P04707 Oryza sativasubsp. indica (Rice) ADH2 Q4R1E8 Solanum lycopersicum (Tomato) ADH2P28032 (Lycopersicon esculentum) Solanum tuberosum (Potato) ADH2 P14674Scheffersomyces stipitis (strain ATCC ADH2, ADH1, O13309 58785/CBS6054/NBRC 10063/NRRL PICST_27980 Y-11545) (Yeast) (Pichia stipitis)Arabidopsis thaliana (Mouse-ear cress) ADH2, ADHIII, Q96533 FDH1,At5g43940, MRH10.4 Saccharomyces cerevisiae (strain ATCC ADH2, ADR2,P00331 204508/S288c) (Baker's yeast) YMR303C, YM9952.05C Candidaalbicans (strain SC5314/ATCC ADH2, O94038 MYA-2876) (Yeast) Ca41C10.04,CaO19.12579, CaO19.5113 Oryza sativa subsp. japonica (Rice) ADH2,DUPR11.1, Q0ITW7 Os11g0210500, LOC_Os11g10510 Drosophila mojavensis(Fruit fly) Adh2, GI17643 P09369 Kluyveromyces lactis (strain ATCC 8585/ADH2, P49383 CBS2359/DSM 70799/NBRC 1267/ KLLA0F18260g NRRL Y-1140/WM37)(Yeast) (Candida sphaerica) Oryctolagus cuniculus (Rabbit) ADH2-1 O46649Oryctolagus cuniculus (Rabbit) ADH2-2 O46650 Hordeum vulgare (Barley)ADH3 P10848 Solanum tuberosum (Potato) ADH3 P14675 Kluyveromyces lactis(strain ATCC 8585/ ADH3, P49384 CBS 2359/DSM 70799/NBRC 1267/KLLA0B09064g NRRL Y-1140/WM37) (Yeast) (Candida sphaerica) Saccharomycescerevisiae (strain ATCC ADH3, P07246 204508/S288c) (Baker's yeast)YMR083W, YM9582.08 Homo sapiens (Human) ADH4 P08319 Mus musculus (Mouse)Adh4 Q9QYY9 Rattus norvegicus (Rat) Adh4 Q64563 Struthio camelus(Ostrich) ADH4 P80468 Kluyveromyces lactis (strain ATCC 8585/ ADH4,P49385 CBS 2359/DSM 70799/NBRC 1267/ KLLA0F13530g NRRL Y-1140/WM37)(Yeast) (Candida sphaerica) Schizosaccharomyces pombe (strain 972/ adh4,Q09669 ATCC 24843) (Fission yeast) SPAC5H10.06c Saccharomyces cerevisiae(strain ADH4, ZRG5, A6ZTT5 YJM789) (Baker's yeast) SCY_1818Saccharomyces cerevisiae (strain ATCC ADH4, ZRG5, P10127 204508/S288c)(Baker's yeast) YGL256W, NRC465 Saccharomyces pastorianus (Lager yeast)ADH5 Q6XQ67 (Saccharomyces cerevisiae × Saccharomyces eubayanus) Bostaurus (Bovine) ADH5 Q3ZC42 Equus caballus (Horse) ADH5 P19854 Musmusculus (Mouse) Adh5, Adh-2, P28474 Adh2 Rattus norvegicus (Rat) Adh5,Adh-2, P12711 Adh2 Oryctolagus cuniculus (Rabbit) ADH5, ADH3 O19053 Homosapiens (Human) ADH5, ADHX, P11766 FDH Dictyostelium discoideum (Slimemold) adh5, Q54TC2 DDB_G0281865 Saccharomyces cerevisiae (strain ATCCADH5, P38113 204508/S288c) (Baker's yeast) YBR145W, YBR1122 Homo sapiens(Human) ADH6 P28332 Peromyscus maniculatus (North American ADH6 P41681deer mouse) Pongo abelii (Sumatran orangutan) (Pongo ADH6 Q5R7Z8pygmaeus abelii) Rattus norvegicus (Rat) Adh6 Q5XI95 Homo sapiens(Human) ADH7 P40394 Rattus norvegicus (Rat) Adh7 P41682 Mus musculus(Mouse) Adh7, Adh-3, Q64437 Adh3 Mycobacterium tuberculosis (strain CDCadhA, MT1911 P9WQC0 1551/Oshkosh) Rhizobium meliloti (strain 1021)(Ensifer adhA, RA0704, O31186 meliloti) (Sinorhizobium meliloti) SMa1296Mycobacterium tuberculosis (strain ATCC adhA, Rv1862 P9WQC1 25618/H37Rv)Zymomonas mobilis subsp. mobilis (strain adhA, ZMO1236 P20368 ATCC31821/ZM4/CP4) Mycobacterium bovis (strain ATCC BAA- adhB, Mb0784cQ7U1B9 935/AF2122/97) Mycobacterium tuberculosis (strain CDC adhB,MT0786 P9WQC6 1551/Oshkosh) Mycobacterium tuberculosis (strain ATCCadhB, Rv0761c, P9WQC7 25618/H37Rv) MTCY369.06c Zymomonas mobilis subsp.mobilis (strain adhB, ZMO1596 P0DJA2 ATCC 31821/ZM4/CP4) Zymomonasmobilis subsp. mobilis (strain adhB, Zmob_1541 F8DVL8 ATCC 10988/DSM424/LMG 404/NCIMB 8938/NRRL B-806/ZM1) Mycobacterium tuberculosis(strain CDC adhD, MT3171 P9WQB8 1551/Oshkosh) Mycobacterium tuberculosis(strain ATCC adhD, Rv3086 P9WQB9 25618/H37Rv) Clostridium acetobutylicum(strain ATCC adhE, aad, P33744 824/DSM 792/JCM 1419/LMG 5710/ CA_P0162VKM B-1787) Escherichia coli (strain K12) adhE, ana, b1241, P0A9Q7JW1228 Escherichia coli O157:H7 adhE, Z2016, P0A9Q8 ECs1741 Rhodobactersphaeroides (strain ATCC adhI, P72324 17023/2.4.1/NCIB 8253/DSM 158)RHOS4_11650, RSP_2576 Oryza sativa subsp. indica (Rice) ADHIII, A2XAZ3OsI_009236 Escherichia coli (strain K12) adhP, yddN, P39451 b1478,JW1474 Geobacillus stearothermophilus (Bacillus adhT P12311stearothermophilus) Emericella nidulans (strain FGSC A4/ alcA, AN8979P08843 ATCC 38163/CBS 112.46/NRRL 194/M139) (Aspergillus nidulans)Emericella nidulans (strain FGSC A4/ alc, AN3741 P54202 ATCC 38163/CBS112.46/NRRL 194/ M139) (Aspergillus nidulans) Emericella nidulans(strain FGSC A4/ alcC, adh3, P07754 ATCC 38163/CBS 112.46/NRRL 194/AN2286 M139) (Aspergillus nidulans) Arabidopsis thaliana (Mouse-earcress) At1g22430, Q95K86 F12K8.22 Arabidopsis thaliana (Mouse-ear cress)At1g22440, Q95K87 F12K8.21 Arabidopsis thaliana (Mouse-ear cress)At1g32780, A1L4Y2 F6N18.16 Arabidopsis thaliana (Mouse-ear cress)At1g64710, Q8VZ49 F13O11.3 Arabidopsis thaliana (Mouse-ear cress)At4g22110, Q0V7W6 F1N20.210 Arabidopsis thaliana (Mouse-ear cress)At5g24760, Q8LEB2 T4C12_30 Arabidopsis thaliana (Mouse-ear cress)At5g42250, Q9FH04 K5J14.5 Zea mays (Maize) FDH P93629 Drosophilamelanogaster (Fruit fly) Fdh, gfd, ODH, P46415 CG6598 Bacillus subtilis(strain 168) gbsB, BSU31050 P71017 Caenorhabditis elegans H24K24.3Q17335 Oryza sativa subsp. japonica (Rice) Os02g0815500, Q0DWH1LOC_Os02g57040, OsJ_008550, P0643F09.4 Mycobacterium tuberculosis(strain Rv1895 O07737 ATCC 25618/H37Rv) Caenorhabditis elegans sodh-1,K12G11.3 Q17334 Caenorhabditis elegans sodh-2, K12G11.4 O45687Pseudomonas sp. terPD P33010 Escherichia coli (strain K12) yiaY, b3589,P37686 JW5648 Moraxella sp. (strain TAE123) P81786 Alligatormississippiensis (American P80222 alligator) Catharanthus roseus(Madagascar P85440 periwinkle) (Vinca rosea) Gadus morhua subsp.callarias (Baltic P26325 cod) (Gadus callarias) Naja naja (Indian cobra)P80512 Pisum sativum (Garden pea) P12886 Pelophylax perezi (Perez'sfrog) (Rana perezi) P22797 Saara hardwickii (Indian spiny-tailed P25405lizard) (Uromastyx hardwickii) Saara hardwickii (Indian spiny-tailedP25406 lizard) (Uromastyx hardwickii) Equus caballus (Horse) P00327Equus caballus (Horse) P00328 Geobacillus stearothermophilus (BacillusP42328 stearothermophilus) Gadus morhua (Atlantic cod) P81600 Gadusmorhua (Atlantic cod) P81601 Myxine glutinosa (Atlantic hagfish) P80360Octopus vulgaris (Common octopus) P81431 Pisum sativum (Garden pea)P80572 Saara hardwickii (Indian spiny-tailed P80467 lizard) (Uromastyxhardwickii) Scyliorhinus canicula (Small-spotted P86884 catshark)(Squalus canicula) Sparus aurata (Gilthead sea bream) P79896

Alcohol Oxidase

The present disclosure describes enzymes that oxidize fatty alcohols tofatty aldehydes.

In some embodiments, an alcohol oxidase (AOX) is used to catalyze theconversion of a fatty alcohol to a fatty aldehyde. Alcohol oxidasescatalyze the conversion of alcohols into corresponding aldehydes (orketones) with electron transfer via the use of molecular oxygen to formhydrogen peroxide as a by-product. AOX enzymes utilize flavin adeninedinucleotide (FAD) as an essential cofactor and regenerate with the helpof oxygen in the reaction medium. Catalase enzymes may be coupled withthe AOX to avoid accumulation of the hydrogen peroxide via catalyticconversion into water and oxygen.

Based on the substrate specificities, AOXs may be categorized into fourgroups: (a) short chain alcohol oxidase, (b) long chain alcohol oxidase,(c) aromatic alcohol oxidase, and (d) secondary alcohol oxidase (Goswamiet al. 2013). Depending on the chain length of the desired substrate,some members of these four groups are better suited than others ascandidates for evaluation.

Short chain alcohol oxidases (including but not limited to thosecurrently classified as EC 1.1.3.13, Table 4) catalyze the oxidation oflower chain length alcohol substrates in the range of C1-C8 carbons (vander Klei et al. 1991) (Ozimek et al. 2005). Aliphatic alcohol oxidasesfrom methylotrophic yeasts such as Candida boidinii and Komagataellapastoris (formerly Pichia pastoris) catalyze the oxidation of primaryalkanols to the corresponding aldehydes with a preference for unbranchedshort-chain aliphatic alcohols. The most broad substrate specificity isfound for alcohol oxidase from the Pichia pastoris including propargylalcohol, 2-chloroethanol, 2-cyanoethanol (Dienys et al. 2003). The majorchallenge encountered in alcohol oxidation is the high reactivity of thealdehyde product. Utilization of a two liquid phase system(water/solvent) can provide in-situ removal of the aldehyde product fromthe reaction phase before it is further converted to the acid. Forexample, hexanal production from hexanol using Pichia pastoris alcoholoxidase coupled with bovine liver catalase was achieved in a bi-phasicsystem by taking advantage of the presence of a stable alcohol oxidasein aqueous phase (Karra-Chaabouni et al. 2003). For example, alcoholoxidase from Pichia pastoris was able to oxidize aliphatic alcohols ofC6 to C11 when used biphasic organic reaction system (Murray and Duff1990). Methods for using alcohol oxidases in a biphasic system accordingto (Karra-Chaabouni et al. 2003) and (Murray and Duff 1990) areincorporated by reference in their entirety.

Long chain alcohol oxidases (including but not limited to thosecurrently classified as EC 1.1.3.20; Table 5) include fatty alcoholoxidases, long chain fatty acid oxidases, and long chain fatty alcoholoxidases that oxidize alcohol substrates with carbon chain length ofgreater than six (Goswami et al. 2013). Banthorpe et al. reported a longchain alcohol oxidase purified from the leaves of Tanacetum vulgare thatwas able to oxidize saturated and unsaturated long chain alcoholsubstrates including hex-trans-2-en-1-ol and octan-1-ol (Banthorpe 1976)(Cardemil 1978). Other plant species, including Simmondsia chinensis(Moreau, R. A., Huang 1979), Arabidopsis thaliana (Cheng et al. 2004),and Lotus japonicas (Zhao et al. 2008) have also been reported assources of long chain alcohol oxidases. Fatty alcohol oxidases aremostly reported from yeast species (Hommel and Ratledge 1990) (Vanhanenet al. 2000) (Hommel et al. 1994) (Kemp et al. 1990) and these enzymesplay an important role in long chain fatty acid metabolism (Cheng et al.2005). Fatty alcohol oxidases from yeast species that degrade and growon long chain alkanes and fatty acid catalyze the oxidation of fattyalcohols. Fatty alcohol oxidase from Candida tropicalis has beenisolated as microsomal cell fractions and characterized for a range ofsubstrates (Eirich et al. 2004) (Kemp et al. 1988) (Kemp et al. 1991)(Mauersberger et al. 1992). Significant activity is observed for primaryalcohols of length C₈ to C₁₆ with reported K_(M) in the 10-50 μM range(Eirich et al. 2004). Alcohol oxidases described may be used for theconversion of medium chain aliphatic alcohols to aldehydes as described,for example, for whole-cells Candida boidinii (Gabelman and Luzio 1997),and Pichia pastoris (Duff and Murray 1988) (Murray and Duff 1990). Longchain alcohol oxidases from filamentous fungi were produced duringgrowth on hydrocarbon substrates (Kumar and Goswami 2006) (Savitha andRatledge 1991). The long chain fatty alcohol oxidase (LjFAO1) from Lotusjaponicas has been heterologously expressed in E. coli and exhibitedbroad substrate specificity for alcohol oxidation including 1-dodecanoland 1-hexadecanol (Zhao et al. 2008).

TABLE 4 Alcohol oxidase enzymes capable of oxidizing short chainalcohols (EC 1.1.3.13) Organism Gene names Accession No. Komagataellapastoris (strain AOX1 F2QY27 ATCC 76273/CBS 7435/CECT PP7435_Chr4-013011047/NRRL Y-11430/Wegner 21-1) (Yeast) (Pichia pastoris) Komagataellapastoris (strain AOX1 P04842 GS115/ATCC 20864) (Yeast) PAS_chr4_0821(Pichia pastoris) Komagataella pastoris (strain AOX2 F2R038 ATCC76273/CBS 7435/CECT PP7435_Chr4-0863 11047/NRRL Y-11430/Wegner 21-1)(Yeast) (Pichia pastoris) Komagataella pastoris (strain AOX2 C4R702GS115/ATCC 20864) (Yeast) PAS_chr4_0152 (Pichia pastoris) Candidaboidinii (Yeast) AOD1 Q00922 Pichia angusta (Yeast) MOX P04841(Hansenula polymorpha) Thanatephorus cucumeris (strain AOD1 BN14_10802M5CC52 AG1-IB/isolate 7/3/14) (Lettuce bottom rot fungus) (Rhizoctoniasolani) Thanatephorus cucumeris (strain MOX BN14_12214 M5CF32AG1-IB/isolate 7/3/14) (Lettuce bottom rot fungus) (Rhizoctonia solani)Thanatephorus cucumeris (strain AOD1 BN14_10691 M5CAV1 AG1-IB/isolate7/3/14) (Lettuce bottom rot fungus) (Rhizoctonia solani) Thanatephoruscucumeris (strain AOD1 BN14_09479 M5C7F4 AG1-IB/isolate 7/3/14) (Lettucebottom rot fungus) (Rhizoctonia solani) Thanatephorus cucumeris (strainAOD1 BN14_10803 M5CB66 AG1-IB/isolate 7/3/14) (Lettuce bottom rotfungus) (Rhizoctonia solani) Thanatephorus cucumeris (strain AOD1BN14_09900 M5C9N9 AG1-IB/isolate 7/3/14) (Lettuce bottom rot fungus)(Rhizoctonia solani) Thanatephorus cucumeris (strain AOD1 BN14_08302M5C2L8 AG1-IB/isolate 7/3/14) (Lettuce bottom rot fungus) (Rhizoctoniasolani) Thanatephorus cucumeris (strain MOX BN14_09408 M5C784AG1-IB/isolate 7/3/14) (Lettuce bottom rot fungus) (Rhizoctonia solani)Thanatephorus cucumeris (strain MOX BN14_09478 M5C8F8 AG1-IB/isolate7/3/14) (Lettuce bottom rot fungus) (Rhizoctonia solani) Thanatephoruscucumeris (strain AOD1 BN14_11356 M5CH40 AG1-IB/isolate 7/3/14) (Lettucebottom rot fungus) (Rhizoctonia solani) Ogataea henricii AOD1 A5LGF0Candida methanosorbosa AOD1 A5LGE5 Candida methanolovescens AOD1 A5LGE4Candida succiphila AOD1 A5LGE6 Aspergillus niger (strain CBS An15g02200A2R501 513.88/FGSC A1513) Aspergillus niger (strain CBS An18g05480A2RB46 513.88/FGSC A1513) Moniliophthora perniciosa I7CMK2(Witches'-broom disease fungus) (Marasmius perniciosus) Candidacariosilignicola AOD1 A5LGE3 Candida pignaliae AOD1 A5LGE1 Candidapignaliae AOD2 A5LGE2 Candida sonorensis AOD1 A5LGD9 Candida sonorensisAOD2 A5LGEO Pichia naganishii AOD1 A5LGF2 Ogataea minuta AOD1 A5LGF1Ogataea philodendra AOD1 A5LGF3 Ogataea wickerhamii AOD1 A5LGE8Kuraishia capsulate AOD1 A5LGE7 Talaromyces stipitatus (strainTSTA_021940 B8MHF8 ATCC 10500/CBS 375.48/QM 6759/NRRL 1006) (Penicilliumstipitatum) Talaromyces stipitatus (strain TSTA_065150 B8LTH7 ATCC10500/CBS 375.48/QM 6759/NRRL 1006) (Penicillium stipitatum) Talaromycesstipitatus (strain TSTA_065150 B8LTH8 ATCC 10500/CBS 375.48/QM 6759/NRRL1006) (Penicillium stipitatum) Talaromyces stipitatus (strainTSTA_000410 B8MSB1 ATCC 10500/CBS 375.48/QM 6759/NRRL 1006) (Penicilliumstipitatum) Ogataea glucozyma AOD1 A5LGE9 Ogataea parapolymorpha (strainHPODL_03886 W1QCJ3 DL-1/ATCC 26012/NRRL Y-7560) (Yeast) (Hansenulapolymorpha) Gloeophyllum trabeum (Brown AOX A8DPS4 rot fungus) Pichiaangusta (Yeast) (Hansenula mox1 A6PZG8 polymorpha) Pichia trehalophilaAOD1 A5LGF4 Pichia angusta (Yeast) (Hansenula mox1 A6PZG9 polymorpha)Pichia angusta (Yeast) (Hansenula mox1 A6PZG7 polymorpha) Ixodesscapularis (Black-legged IscW_ISCW017898 B7PIZ7 tick) (Deer tick)

TABLE 5 Alcohol oxidase enzymes capable of oxidizing long chain alcoholsincluding fatty alcohols (EC 1.1.3.20) Organism Gene names Accession No.Lotus japonicus (Lotus corniculatus var. FAO1 B5WWZ8 japonicus)Arabidopsis thaliana (Mouse-ear cress) FAO1 At1g03990 F21M11.7 Q9ZWB9Lotus japonicus (Lotus corniculatus var. FAO2 B5WWZ9 japonicus)Arabidopsis thaliana (Mouse-ear cress) FAO3 At3g23410 MLM24.14 Q9LW56MLM24.23 Arabidopsis thaliana (Mouse-ear cress) FAO4A At4g19380 O65709T5K18.160 Arabidopsis thaliana (Mouse-ear cress) FAO4B At4g28570T5F17.20 Q94BP3 Microbotryum violaceum (strain p1A1 MVLG_06864 U5HIL4Lamole) (Anther smut fungus) (Ustilago violacea) Ajellomycesdermatitidis ATCC 26199 BDFG_03507 T5BNQ0 Gibberella zeae (strainPH-1/ATCC FG06918.1 FGSG_06918 I1RS14 MYA-4620/FGSC 9075/NRRL 31084)(Wheat head blight fungus) (Fusarium graminearum) Pichia sorbitophila(strain ATCC MYA- Piso0_004410 G8Y5E1 4447/BCRC 22081/CBS 7064/GNLVRS01_PISO0K16268g NBRC 10061/NRRL Y-12695) GNLVRS01_PISO0L16269g(Hybrid yeast) Emericella nidulans (strain FGSC A4/ AN0623.2 ANIA_00623Q5BFQ7 ATCC 38163/CBS 112.46/NRRL 194/ M139) (Aspergillus nidulans)Pyrenophora tritici-repentis (strain Pt- PTRG_10154 B2WJW5 1C-BFP)(Wheat tan spot fungus) (Drechslera tritici-repentis) Paracoccidioideslutzii (strain ATCC PAAG_09117 C1HEC6 MYA-826/Pb01) (Paracoccidioidesbrasiliensis) Candida parapsilosis (strain CDC 317/ CPAR2_204420 G8BG15ATCC MYA-4646) (Yeast) (Monilia parapsilosis) Pseudozyma brasiliensis(strain PSEUBRA_SCAF2g03010 V5GPS6 GHG001) (Yeast) Candida parapsilosis(strain CDC 317/ CPAR2_204430 G8BG16 ATCC MYA-4646) (Yeast) (Moniliaparapsilosis) Sclerotinia borealis F-4157 SBOR_5750 W9CDE2 Sordariamacrospora (strain ATCC SMAC_06361 F7W6K4 MYA-333/DSM 997/K(L3346)/K-hell) Sordaria macrospora (strain ATCC SMAC_01933 F7VSA1 MYA-333/DSM997/K(L3346)/K- hell) Meyerozyma guilliermondii (strain PGUG_03467A5DJL6 ATCC 6260/CBS 566/DSM 6381/ JCM 1539/NBRC 10279/NRRL Y- 324)(Yeast) (Candida guilliermondii) Trichophyton rubrum CBS 202.88H107_00669 A0A023ATC5 Arthrobotrys oligospora (strain ATCCAOL_s00097g516 G1XJI9 24927/CBS 115.81/DSM 1491) (Nematode-trappingfungus) (Didymozoophaga oligospora) Scheffersomyces stipitis (strainATCC FAO1 PICST_90828 A3LYX9 58785/CBS 6054/NBRC 10063/ NRRL Y-11545)(Yeast) (Pichia stipitis) Scheffersomyces stipitis (strain ATCC FAO2PICST_32359 A3LW61 58785/CBS 6054/NBRC 10063/ NRRL Y-11545) (Yeast)(Pichia stipitis) Aspergillus oryzae (strain 3.042) Ao3042_09114 I8TL25(Yellow koji mold) Fusarium oxysporum (strain Fo5176) FOXB_17532 F9GFU8(Fusarium vascular wilt) Rhizopus delemar (strain RA 99-880/ RO3G_08271I1C536 ATCC MYA-4621/FGSC 9543/ NRRL 43880) (Mucormycosis agent)(Rhizopus arrhizus var. delemar) Rhizopus delemar (strain RA 99-880/RO3G_00154 I1BGX0 ATCC MYA-4621/FGSC 9543/ NRRL 43880) (Mucormycosisagent) (Rhizopus arrhizus var. delemar) Fusarium oxysporum (strainFo5176) FOXB_07532 F9FMA2 (Fusarium vascular wilt) Penicilliumroqueforti PROQFM164_S02g001772 W6QPY1 Aspergillus clavatus (strain ATCC1007/ ACLA_018400 A1CNB5 CBS 513.65/DSM 816/NCTC 3887/ NRRL 1)Arthroderma otae (strain ATCC MYA- MCYG_08732 C5G1B0 4605/CBS 113480)(Microsporum canis) Trichophyton tonsurans (strain CBS TESG_07214 F2S8I2112818) (Scalp ringworm fungus) Colletotrichum higginsianum (strain IMICH063_13441 H1VUE7 349063) (Crucifer anthracnose fungus) Ajellomycescapsulatus (strain H143) HCDG_07658 C6HN77 (Darling's disease fungus)(Histoplasma capsulatum) Trichophyton rubrum (strain ATCC TERG_08235F2T096 MYA-4607/CBS 118892) (Athlete's foot fungus) Cochliobolusheterostrophus (strain C5/ COCHEDRAFT_1201414 M2UMT9 ATCC 48332/race O)(Southern corn leaf blight fungus) (Bipolaris maydis) Candidaorthopsilosis (strain 90-125) CORT_0D04510 H8X643 (Yeast) Candidaorthopsilosis (strain 90-125) CORT_0D04520 H8X644 (Yeast) Candidaorthopsilosis (strain 90-125) CORT_0D04530 H8X645 (Yeast) Pseudozymaaphidis DSM 70725 PaG_03027 W3VP49 Coccidioides posadasii (strain C735)CPC735_000380 C5P005 (Valley fever fungus) Magnaporthe oryzae (strainP131) (Rice OOW_P131scaffold01214g15 L7IZ92 blast fungus) (Pyriculariaoryzae) Neurospora tetrasperma (strain FGSC NEUTE1DRAFT_82541 F8MKD12508/ATCC MYA-4615/P0657) Hypocrea virens (strain Gy29-8/FGSCTRIVIDRAFT_54537 G9MMY7 10586) (Gliocladium virens) (Trichoderma virens)Hypocrea virens (strain Gy29-8/FGSC TRIVIDRAFT_53801 G9MT89 10586)(Gliocladium virens) (Trichoderma virens) Aspergillus niger (strain CBS513.88/ An01g09620 A2Q9Z3 FGSC A1513) Verticillium dahliae (strainVdLs.17/ VDAG_05780 G2X6J8 ATCC MYA-4575/FGSC 10137) (Verticillium wilt)Ustilago maydis (strain 521/FGSC UM02023.1 Q4PCZ0 9021) (Corn smutfungus) Fusarium oxysporum f. sp. lycopersici FOWG_13006 W9LNI9 MN25Fusarium oxysporum f. sp. lycopersici FOWG_02542 W9N9Z1 MN25 Candidatropicalis (Yeast) FAO1 Q6QIR6 Magnaporthe oryzae (strain 70-15/MGG_11317 G4MVK1 ATCC MYA-4617/FGSC 8958) (Rice blast fungus)(Pyricularia oryzae) Candida tropicalis (Yeast) faot Q9P8D9 Candidatropicalis (Yeast) FAO2a Q6QIR5 Phaeosphaeria nodorum (strain SN15/SNOG_02371 Q0V0U3 ATCC MYA-4574/FGSC 10173) (Glume blotch fungus)(Septoria nodorum) Candida tropicalis (Yeast) FAO2b Q6QIR4Pestalotiopsis fici W106-1 PFICI_11209 W3WU04 Magnaporthe oryzae (strainY34) (Rice OOU_Y34scaffold00240g57 L7IFT5 blast fungus) (Pyriculariaoryzae) Pseudogymnoascus destructans (strain GMDG_01756 L8G0G6 ATCCMYA-4855/20631-21) (Bat white-nose syndrome fungus) (Geomycesdestructans) Pseudogymnoascus destructans (strain GMDG_04950 L8GCY2 ATCCMYA-4855/20631-21) (Bat white-nose syndrome fungus) (Geomycesdestructans) Mycosphaerella fijiensis (strain MYCFIDRAFT_52380 M2Z831CIRAD86) (Black leaf streak disease fungus) (Pseudocercospora fijiensis)Bipolaris oryzae ATCC 44560 COCMIDRAFT_84580 W7A0I8 Cladophialophorapsammophila CBS A1O5_08147 W9WTM9 110553 Fusarium oxysporum f. sp.melonis FOMG_05173 X0AEE6 26406 Fusarium oxysporum f. sp. melonisFOMG_17829 W9ZBB7 26406 Cyphellophora europaea CBS 101466HMPREF1541_02174 W2S2S5 Aspergillus kawachii (strain NBRC AKAW_00147G7X626 4308) (White koji mold) (Aspergillus awamori var. kawachi)Aspergillus terreus (strain NIH 2624/ ATEG_05086 Q0CMJ8 FGSC A1156)Coccidioides immitis (strain RS) (Valley CIMG_02987 J3KAI8 fever fungus)Ajellomyces dermatitidis (strain ER-3/ BDCG_04701 C5GLS5 ATCC MYA-2586)(Blastomyces dermatitidis) Fusarium oxysporum f. sp. cubenseFOC1_g10013865 N4U732 (strain race 1) (Panama disease fungus)Rhodotorula glutinis (strain ATCC RTG_00643 G0SVU8 204091/IIP 30/MTCC1151) (Yeast) Aspergillus niger (strain ATCC 1015/ ASPNIDRAFT_35778G3XTM6 CBS 113.46/FGSC A1144/LSHB Ac4/ NCTC 3858a/NRRL 328/USDA 3528.7)Candida cloacae fao1 Q9P8D8 Candida cloacae fao2 Q9P8D7 Fusariumoxysporum f. sp. cubense FOC1_10006358 N4TUH3 (strain race 1) (Panamadisease fungus) Candida albicans (strain SC5314/ FAO1 CaO19.13562 Q59RS8ATCC MYA-2876) (Yeast) orf19.13562 Candida albicans (strain SC5314/ FAO1CaO19.6143 Q59RP0 ATCC MYA-2876) (Yeast) orf19.6143 Chaetomiumthermophilum (strain DSM CTHT_0018560 G0S2U9 1495/CBS 144.50/IMI 039719)Mucor circinelloides f. circinelloides HMPREF1544_05296 S2JDN0 (strain1006PhL) (Mucormycosis agent) (Calyptromyces circinelloides) Mucorcircinelloides f. circinelloides HMPREF1544_05295 S2JYP5 (strain1006PhL) (Mucormycosis agent) (Calyptromyces circinelloides) Mucorcircinelloides f. circinelloides HMPREF1544_06348 S2JVK9 (strain1006PhL) (Mucormycosis agent) (Calyptromyces circinelloides) Botryotiniafuckeliana (strain BcDW1) BcDW1_6807 M7UD26 (Noble rot fungus) (Botrytiscinerea) Podospora anserina (strain S/ATCC PODANS_5_13040 B2AFD8MYA-4624/DSM 980/FGSC 10383) (Pleurage anserina) Neosartorya fumigata(strain ATCC AFUA_1G17110 Q4WR91 MYA-4609/Af293/CBS 101355/ FGSC A1100)(Aspergillus fumigatus) Fusarium oxysporum f. sp. vasinfectum FOTG_00686X0MEE6 25433 Fusarium oxysporum f. sp. vasinfectum FOTG_12485 X0LE9825433 Trichophyton interdigitale H6 H101_06625 A0A022U717 Beauveriabassiana (strain ARSEF 2860) BBA_04100 J4UNY3 (White muscardine diseasefungus) (Tritirachium shiotae) Fusarium oxysporum f. sp. radicis-FOCG_00843 X0GQ62 lycopersici 26381 Fusarium oxysporum f. sp. radicis-FOCG_15170 X0F4T1 lycopersici 26381 Neurospora tetrasperma (strain FGSCNEUTE2DRAFT_88670 G4UNN6 2509/P0656) Pseudozyma hubeiensis (strain SY62)PHSY_000086 R9NVU1 (Yeast) Lodderomyces elongisporus (strain LELG_03289A5E102 ATCC 11503/CBS 2605/JCM 1781/ NBRC 1676/NRRL YB-4239) (Yeast)(Saccharomyces elongisporus) Malassezia globosa (strain ATCC MYA-MGL_3855 A8QAY8 4612/CBS 7966) (Dandruff-associated fungus) Byssochlamysspectabilis (strain No. 5/ PVAR5_7014 V5GBL6 NBRC 109023) (Paecilomycesvariotii) Ajellomyces capsulatus (strain H88) HCEG_03274 F0UF47(Darling's disease fungus) (Histoplasma capsulatum) Trichosporon asahiivar. asahii (strain A1Q1_03669 J6FBP4 ATCC 90039/CBS 2479/JCM 2466/ KCTC7840/NCYC 2677/UAMH 7654) (Yeast) Penicillium oxalicum (strain 114-2/PDE_00027 S7Z8U8 CGMCC 5302) (Penicillium decumbens) Fusarium oxysporumf. sp. conglutinans FOPG_02304 X0IBE3 race 2 54008 Fusarium oxysporum f.sp. conglutinans FOPG_13066 X0H540 race 2 54008 Fusarium oxysporum f.sp. raphani FOQG_00704 X0D1G8 54005 Fusarium oxysporum f. sp. raphaniFOQG_10402 X0C482 54005 Metarhizium acridum (strain CQMa MAC_03115E9DZR7 102) Arthroderma benhamiae (strain ATCC ARB_02250 D4B1C1MYA-4681/CBS 112371) (Trichophyton mentagrophytes) Fusarium oxysporum f.sp. cubense FOIG_12161 X0JFI6 tropical race 4 54006 Fusarium oxysporumf. sp. cubense FOIG_12751 X0JDU5 tropical race 4 54006 Cochliobolusheterostrophus (strain C4/ COCC4DRAFT_52836 N4WZZ0 ATCC 48331/race T)(Southern corn leaf blight fungus) (Bipolaris maydis) Trichosporonasahii var. asahii (strain A1Q2_00631 K1VZW1 CBS 8904) (Yeast)Mycosphaerella graminicola (strain CBS MYCGRDRAFT_37086 F9X375115943/IPO323) (Speckled leaf blotch fungus) (Septoria tritici)Botryotinia fuckeliana (strain T4) BofuT4_P072020.1 G2XQ18 (Noble rotfungus) (Botrytis cinerea) Metarhizium anisopliae (strain ARSEFMAA_05783 E9F0I4 23/ATCC MYA-3075) Cladophialophora carrionii CBS 160.54G647_05801 V9DAR1 Coccidioides posadasii (strain RMSCC CPSG_09174 E9DH75757/Silveira) (Valley fever fungus) Rhodosporidium toruloides (strainRHTO_06879 M7X159 NP11) (Yeast) (Rhodotorula gracilis) Puccinia graminisf. sp. tritici (strain PGTG_10521 E3KIL8 CRL 75-36-700-3/race SCCL)(Black stem rust fungus) Trichophyton rubrum CBS 288.86 H103_00624A0A022WG28 Colletotrichum fioriniae PJ7 CFIO01_08202 A0A010RKZ4Trichophyton rubrum CBS 289.86 H104_00611 A0A022XB46 Cladophialophorayegresii CBS 114405 A1O7_02579 W9WC55 Colletotrichum orbiculare (strain104-T/ Cob_10151 N4VFP3 ATCC 96160/CBS 514.97/LARS 414/ MAFF 240422)(Cucumber anthracnose fungus) (Colletotrichum lagenarium) Drechslerellastenobrocha 248 DRE_03459 W7IDL6 Neosartorya fumigata (strain CEA10/AFUB_016500 B0XP90 CBS 144.89/FGSC A1163) (Aspergillus fumigatus)Thielavia terrestris (strain ATCC 38088/ THITE_2117674 G2R8H9 NRRL 8126)(Acremonium alabamense) Gibberella fujikuroi (strain CBS 195.34/FFUJ_02948 S0DZP7 IMI 58289/NRRL A-6831) (Bakanae and foot rot diseasefungus) (Fusarium fujikuroi) Gibberella fujikuroi (strain CBS 195.34/FFUJ_12030 S0EMC6 IMI 58289/NRRL A-6831) (Bakanae and foot rot diseasefungus) (Fusarium fujikuroi) Aspergillus flavus (strain ATCC 200026/AFLA_109870 B8N941 FGSC A1120/NRRL 3357/JCM 12722/SRRC 167) Togniniaminima (strain UCR-PA7) UCRPA7_1719 R8BTZ6 (Esca disease fungus)(Phaeoacremonium aleophilum) Ajellomyces dermatitidis (strain ATCCBDDG_09783 F2TUC0 18188/CBS 674.68) (Blastomyces dermatitidis)Macrophomina phaseolina (strain MS6) MPH_10582 K2RHA5 (Charcoal rotfungus) Neurospora crassa (strain ATCC 24698/ NCU08977 Q7S2Z274-OR23-1A/CBS 708.71/DSM 1257/ FGSC 987) Neosartorya fischeri (strainATCC 1020/ NFIA_008260 A1D156 DSM 3700/FGSC A1164/NRRL 181) (Aspergillusfischerianus) Fusarium pseudograminearum (strain FPSE_11742 K3U9J5 C53096) (Wheat and barley crown-rot fungus) Spathaspora passalidarum(strain NRRL SPAPADRAFT_54193 G3AJP0 Y-27907/11-Y1) Spathasporapassalidarum (strain NRRL SPAPADRAFT_67198 G3ANX7 Y-27907/11-Y1)Trichophyton verrucosum (strain HKI TRV_07960 D4DL86 0517) Arthrodermagypseum (strain ATCC MGYG_07264 E4V2J0 MYA-4604/CBS 118893) (Microsporumgypseum) Hypocrea jecorina (strain QM6a) TRIREDRAFT_43893 G0R7P8(Trichoderma reesei) Trichophyton rubrum MR1448 H110_00629 A0A022Z1G4Aspergillus ruber CBS 135680 EURHEDRAFT_512125 A0A017SPR0 Glarealozoyensis (strain ATCC 20868/ GLAREA_04397 S3D6C1 MF5171) Setosphaeriaturcica (strain 28A) SETTUDRAFT_20639 R0K6H8 (Northern leaf blightfungus) (Exserohilum turcicum) Paracoccidioides brasiliensis (strainPADG_06552 C1GH16 Pb18) Fusarium oxysporum Fo47 FOZG_13577 W9JPG9Fusarium oxysporum Fo47 FOZG_05344 W9KPH3 Trichophyton rubrum MR1459H113_00628 A0A022ZY09 Penicillium marneffei (strain ATCC PMAA_075740B6QBY3 18224/CBS 334.59/QM 7333) Sphaerulina musiva (strain SO2202)SEPMUDRAFT_154026 M3DAK6 (Poplar stem canker fungus) (Septoria musiva)Gibberella moniliformis (strain M3125/ FVEG_10526 W7N4P8 FGSC 7600)(Maize ear and stalk rot fungus) (Fusarium verticillioides) Gibberellamoniliformis (strain M3125/ FVEG_08281 W7MVR9 FGSC 7600) (Maize ear andstalk rot fungus) (Fusarium verticillioides) Pseudozyma antarctica(strain T-34) PANT_22d00298 M9MGF2 (Yeast) (Candida antarctica)Paracoccidioides brasiliensis (strain PABG_07795 C0SJD4 Pb03)Rhizophagus irregularis (strain DAOM GLOINDRAFT_82554 U9TF61 181602/DAOM197198/MUCL 43194) (Arbuscular mycorrhizal fungus) (Glomus intraradices)Penicillium chrysogenum (strain ATCC Pc21g23700 B6HJ58 28089/DSM1075/Wisconsin 54- PCH_Pc21g23700 1255) (Penicillium notatum) Baudoiniacompniacensis (strain UAMH BAUCODRAFT_274597 M2M6Z5 10762) (Angels'share fungus) Hypocrea atroviridis (strain ATCC TRIATDRAFT_280929 G9NJ3220476/IMI 206040) (Trichoderma atroviride) Colletotrichumgloeosporioides (strain CGLO_06642 T0LPH0 Cg-14) (Anthracnose fungus)(Glomerella cingulata) Cordyceps militaris (strain CM01) CCM_02665G3JB34 (Caterpillar fungus) Pyronema omphalodes (strain CBS PCON_13062U4LKE9 100304) (Pyronema confluens) Colletotrichum graminicola (strainGLRG_08499 E3QR67 M1.001/M2/FGSC 10212) (Maize anthracnose fungus)(Glomerella graminicola) Glarea lozoyensis (strain ATCC 74030/ M7I_2117H0EHX4 MF5533) Fusarium oxysporum f. sp. cubense FOC4_g 10002493 N1S969(strain race 4) (Panama disease fungus) Fusarium oxysporum f. sp.cubense FOC4_10011461 N1RT80 (strain race 4) (Panama disease fungus)Cochliobolus sativus (strain ND90Pr/ COCSADRAFT_295770 M2TBE4 ATCC201652) (Common root rot and spot blotch fungus) (Bipolaris sorokiniana)Mixia osmundae (strain CBS 9802/ Mo05571 E5Q_05571 G7E7S3 IAM 14324/JCM22182/KY 12970) Mycosphaerella pini (strain NZE10/ DOTSEDRAFT_69651N1PXR0 CBS 128990) (Red band needle blight fungus) (Dothistromaseptosporum) Grosmarmia clavigera (strain kw1407/ CMQ_1113 F0XC64 UAMH11150) (Blue stain fungus) (Graphiocladiella clavigera) Fusariumoxysporum FOSC 3-a FOYG_03004 W9IUE5 Fusarium oxysporum FOSC 3-aFOYG_16040 W9HNP0 Fusarium oxysporum FOSC 3-a FOYG_17058 W9HB31 Nectriahaematococca (strain 77-13-4/ NECHADRAFT_37686 C7YQL1 ATCC MYA-4622/FGSC9596/ MPVI) (Fusarium solani subsp. pisi) Nectria haematococca (strain77-13-4/ NECHADRAFT_77262 C7ZJI0 ATCC MYA-4622/FGSC 9596/ MPVI)(Fusarium solani subsp. pisi) Tuber melanosporum (strain Mel28)GSTUM_00010376001 D5GLS0 (Perigord black truffle) Ajellomycesdermatitidis (strain BDBG_07633 C5JYI9 SLH14081) (Blastomycesdermatitidis) Chaetomium globosum (strain ATCC CHGG_09885 Q2GQ696205/CBS 148.51/DSM 1962/NBRC 6347/NRRL 1970) (Soil fungus) Candidatenuis (strain ATCC 10573/ CANTEDRAFT_108652 G3B9Z1 BCRC 21748/CBS615/JCM 9827/ NBRC 10315/NRRL Y-1498/VKM Y-70) (Yeast) Trichophytonrubrum CBS 100081 H102_00622 A0A022VKY4 Pyrenophora teres f. teres(strain 0-1) PTT_09421 E3RLZ3 (Barley net blotch fungus) (Drechslerateres f. teres) Colletotrichum gloeosporioides (strain CGGC5_4608 L2GB29Nara gc5) (Anthracnose fungus) (Glomerella cingulata) Gibberella zeae(Wheat head blight FG05_06918 A0A016PCS4 fungus) (Fusarium graminearum)Trichophyton soudanense CBS 452.61 H105_00612 A0A022Y6A6 Sclerotiniasclerotiorum (strain ATCC SS1G_07437 A7EQ37 18683/1980/Ss-1) (Whitemold) (Whetzelinia sclerotiorum) Fusarium oxysporum f. sp. pisi HDV247FOVG_14401 W9NWU8 Fusarium oxysporum f. sp. pisi HDV247 FOVG_02874W9Q5V3 Ustilago hordei (strain Uh4875-4) UHOR_03009 I2G1Z4 (Barleycovered smut fungus) Sporisorium reilianum (strain SRZ2) sr12985 E6ZYF7(Maize head smut fungus) Bipolaris zeicola 26-R-13 COCCADRAFT_81154W6YIP8 Melampsora larici-populina (strain MELLADRAFT_78490 F4RUZ898AG31/pathotype 3-4-7) (Poplar leaf rust fungus) Fusarium oxysporum f.sp. lycopersici FOXG_01901 J9MG95 (strain 4287/CBS 123668/FGSC 9935/NRRL 34936) (Fusarium vascular wilt of tomato) Fusarium oxysporum f. sp.lycopersici FOXG_11941 J9N9S4 (strain 4287/CBS 123668/FGSC 9935/ NRRL34936) (Fusarium vascular wilt of tomato) Bipolaris victoriae FI3COCVIDRAFT_39053 W7EMJ8 Debaryomyces hansenii (strain ATCC DEHA2E04268gQ6BQL4 36239/CBS 767/JCM 1990/NBRC 0083/IGC 2968) (Yeast) (Torulasporahansenii) Clavispora lusitaniae (strain ATCC CLUG_01505 C4XZX3 42720)(Yeast) (Candida lusitaniae) Candida albicans (strain WO-1) (Yeast)CAWG_02023 C4YME4 Trichophyton rubrum MR850 H100_00625 A0A022U0Q2Candida dubliniensis (strain CD36/ CD36_32890 B9WMC7 ATCC MYA-646/CBS7987/NCPF 3949/NRRL Y-17841) (Yeast) Starmerella bombicola AOX1A0A024FB95 Thielavia heterothallica (strain ATCC MYCTH_103590 G2QJL742464/BCRC 31852/DSM 1799) (Myceliophthora thermophila) Clavicepspurpurea (strain 20.1) (Ergot CPUR_07614 M1WFI4 fungus) (Sphaceliasegetum) Aspergillus oryzae (strain ATCC 42149/ AO090023000571 Q2UH61RIB 40) (Yellow koji mold) Dictyostelium discoideum (Slime mold)DDB_0184181 Q54DT6 DDB_G0292042 Triticum urartu (Red wild einkorn)TRIUR3_22733 M7YME5 (Crithodium urartu) Solanum tuberosum (Potato)PGSC0003DMG400017211 M1BG07 Oryza sativa subsp. japonica (Rice)OSJNBb0044B19.5 Q8W5P8 LOC_Os10g33540 Oryza sativa subsp. japonica(Rice) OJ1234_B11.20 Q6K9N5 Os02g0621800 Oryza sativa subsp. japonica(Rice) OSJNBa0001K12.5 Q8W5P3 LOC_Os10g33520 Zea mays (Maize)ZEAMMB73_809149 C0P3J6 Citrus clementina CICLE_v10011111mg V4S9P4 Citrusclementina CICLE_v10018992mg V4U4C9 Citrus clementina CICLE_v10004405mgV4S9D3 Citrus clementina CICLE_v10004403mg V4RZZ6 Morus notabilisL484_011703 W9RIK0 Morus notabilis L484_005930 W9RET7 Medicagotruncatula (Barrel medic) MTR_1g075650 G7I4U3 (Medicago tribuloides)Arabidopsis thaliana (Mouse-ear cress) Q8LDP0 Medicago truncatula(Barrel medic) MTR_4g081080 G7JF07 (Medicago tribuloides) Simmondsiachinensis (Jojoba) (Buxus L7VFV2 chinensis) Prunus persica (Peach)(Amygdalus PRUPE_ppa018458mg M5VXL1 persica) Aphanomyces astaciH257_07411 W4GI89 Aphanomyces astaci H257_07412 W4GI44 Aphanomycesastaci H257_07411 W4GKE3 Aphanomyces astaci H257_07411 W4GK29Aphanomyces astaci H257_07411 W4GJ79 Aphanomyces astaci H257_07411W4GI38 Phaeodactylum tricornutum (strain PHATRDRAFT_48204 B7G6C1 CCAP1055/1) Hordeum vulgare var. distichum (Two- F2E4R4 rowed barley)Hordeum vulgare var. distichum (Two- F2DZG1 rowed barley) Hordeumvulgare var. distichum (Two- M0YPG7 rowed barley) Hordeum vulgare var.distichum (Two- M0YPG6 rowed barley) Hordeum vulgare var. distichum(Two- F2CUY4 rowed barley) Ricinus communis (Castor bean) RCOM_0867830B9S1S3 Brassica rapa subsp. pekinensis (Chinese BRA014947 M4DEM5cabbage) (Brassica pekinensis) Ricinus communis (Castor bean)RCOM_0258730 B9SV13 Brassica rapa subsp. pekinensis (Chinese BRA001912M4CCI2 cabbage) (Brassica pekinensis) Brassica rapa subsp. pekinensis(Chinese BRA012548 M4D7T8 cabbage) (Brassica pekinensis) Brassica rapasubsp. pekinensis (Chinese BRA024190 M4E5Y6 cabbage) (Brassicapekinensis) Brassica rapa subsp. pekinensis (Chinese BRA015283 M4DFL0cabbage) (Brassica pekinensis) Ricinus communis (Castor bean)RCOM_1168730 B9SS54 Zea mays (Maize) C4J691 Oryza glaberrima (Africanrice) I1P2B7 Zea mays (Maize) B6SXM3 Zea mays (Maize) C0HFU4 Aegilopstauschii (Tausch's goatgrass) F775_9577 R7W4J3 (Aegilops squarrosa)Solanum habrochaites (Wild tomato) R9R6T0 (Lycopersicon hirsutum)Physcomitrella patens subsp. patens PHYPADRAFT_124285 A9S535 (Moss)Physcomitrella patens subsp. patens PHYPADRAFT_113581 A9RG13 (Moss)Physcomitrella patens subsp. patens PHYPADRAFT_182504 A9S9A5 (Moss)Solanum pennellii (Tomato) R9R6Q1 (Lycopersicon pennellii) Vitisvinifera (Grape) VIT_02s0087g00630 F6HJ27 Vitis vinifera (Grape)VIT_07s0005g03780 F6HZM3 Vitis vinifera (Grape) VIT_05s0049g01400 F6H8T4Vitis vinifera (Grape) VITISV_019349 A5AH38 Capsella rubellaCARUB_v10013046mg R0HIT3 Capsella rubella CARUB_v10004212mg R0GUX4Capsella rubella CARUB_v10004208mg R0F3X6 Capsella rubellaCARUB_v10012453mg R0ILD0 Capsella rubella CARUB_v10004208mg R0GUX1Eutrema salsugineum (Saltwater cress) EUTSA_v10024496mg V4MD54(Sisymbrium salsugineum) Eutrema salsugineum (Saltwater cress)EUTSA_v10020141mg V4NM59 (Sisymbrium salsugineum) Eutrema salsugineum(Saltwater cress) EUTSA_v10024496mg V4LUR9 (Sisymbrium salsugineum)Eutrema salsugineum (Saltwater cress) EUTSA_v10024528mg V4P767(Sisymbrium salsugineum) Eutrema salsugineum (Saltwater cress)EUTSA_v10006882mg V4L2P6 (Sisymbrium salsugineum) Selaginellamoellendorffii (Spikemoss) SELMODRAFT_87684 D8R6Z6 Selaginellamoellendorffii (Spikemoss) SELMODRAFT_87621 D8R6Z5 Selaginellamoellendorffii (Spikemoss) SELMODRAFT_74601 D8QN81 Selaginellamoellendorffii (Spikemoss) SELMODRAFT_73531 D8QN82 Sorghum bicolor(Sorghum) (Sorghum Sb04g026390 C5XXS4 vulgare) SORBIDRAFT_04g026390Sorghum bicolor (Sorghum) (Sorghum Sb04g026370 C5XXS1 vulgare)SORBIDRAFT_04g026370 Sorghum bicolor (Sorghum) (Sorghum Sb01g019470C5WYH6 vulgare) SORBIDRAFT_01g019470 Sorghum bicolor (Sorghum) (SorghumSb01g019480 C5WYH7 vulgare) SORBIDRAFT_01g019480 Sorghum bicolor(Sorghum) (Sorghum Sb01g019460 C5WYH5 vulgare) SORBIDRAFT_01g019460Solanum pimpinellifolium (Currant R9R6J2 tomato) (Lycopersiconpimpinellifolium) Phaseolus vulgaris (Kidney bean) PHAVU_007G124200gV7BGM7 (French bean) Phaseolus vulgaris (Kidney bean) PHAVU_011G136600gV7AI35 (French bean) Phaseolus vulgaris (Kidney bean) PHAVU_001G162800gV7D063 (French bean) Solanum tuberosum (Potato) PGSC0003DMG400024294M1C923 Solanum tuberosum (Potato) PGSC0003DMG400018458 M1BKV4 Solanumtuberosum (Potato) PGSC0003DMG400018458 M1BKV3 Glycine max (Soybean)(Glycine K7LK61 hispida) Glycine max (Soybean) (Glycine K7KXQ9 hispida)Populus trichocarpa (Western balsam POPTR_0008s16920g B9HKS3 poplar)(Populus balsamifera subsp. trichocarpa) Picea sitchensis (Sitka spruce)(Pinus B8LQ84 sitchensis) Populus trichocarpa (Western balsamPOPTR_0004s24310g U5GKQ5 poplar) (Populus balsamifera subsp.trichocarpa) Populus trichocarpa (Western balsam POPTR_0010s07980gB9HSG9 poplar) (Populus balsamifera subsp. trichocarpa) Glycine max(Soybean) (Glycine I1N9S7 hispida) Glycine max (Soybean) (Glycine I1LSK5hispida) Setaria italica (Foxtail millet) (Panicum Si034362m.g K4A658italicum) Solanum lycopersicum (Tomato) Solyc09g072610.2 K4CUT7(Lycopersicon esculentum) Setaria italica (Foxtail millet) (PanicumSi016380m.g K3YQ38 italicum) Solanum lycopersicum (Tomato) R9R6I9(Lycopersicon esculentum) Solanum lycopersicum (Tomato) Solyc09g090350.2K4CW61 (Lycopersicon esculentum) Solanum lycopersicum (Tomato)Solyc08g005630.2 K4CI54 (Lycopersicon esculentum) Solanum lycopersicum(Tomato) Solyc08g075240.2 K4CMP1 (Lycopersicon esculentum) Setariaitalica (Foxtail millet) (Panicum Si034359m.g K4A655 italicum) Setariaitalica (Foxtail millet) (Panicum Si034354m.g K4A650 italicum) Mimulusguttatus (Spotted monkey MIMGU_mgv1a001896mg A0A022PU07 flower) (Yellowmonkey flower) Mimulus guttatus (Spotted monkey MIMGU_mgv1a022390mgA0A022RAV4 flower) (Yellow monkey flower) Mimulus guttatus (Spottedmonkey MIMGU_mgv1a001868mg A0A022S2E6 flower) (Yellow monkey flower)Mimulus guttatus (Spotted monkey MIMGU_mgv1a001883mg A0A022S275 flower)(Yellow monkey flower) Mimulus guttatus (Spotted monkeyMIMGU_mgv1a001761mg A0A022QNF0 flower) (Yellow monkey flower) Musaacuminata subsp. malaccensis M0SNA8 (Wild banana) (Musa malaccensis)Musa acuminata subsp. malaccensis M0RUT7 (Wild banana) (Musamalaccensis) Musa acuminata subsp. malaccensis M0RUK3 (Wild banana)(Musa malaccensis) Saprolegnia diclina VS20 SDRG_10901 T0RG89Brachypodium distachyon (Purple false BRADI3G49085 I1IBP7 brome)(Trachynia distachya) Brachypodium distachyon (Purple false BRADI3G28677I1I4N2 brome) (Trachynia distachya) Brachypodium distachyon (Purplefalse BRADI3G28657 I1I4N0 brome) (Trachynia distachya) Oryza sativasubsp. indica (Rice) OsI_34012 B8BHG0 Oryza sativa subsp. indica (Rice)OsI_08118 B8AFT8 Oryza sativa subsp. indica (Rice) OsI_34008 A2Z8H1Oryza sativa subsp. indica (Rice) OsI_34014 B8BHG1 Oryza sativa subsp.japonica (Rice) LOC_Os10g33460 Q7XDG3 Oryza sativa subsp. japonica(Rice) Os10g0474800 Q0IX12 Oryza sativa subsp. japonica (Rice)Os10g0474966 C7J7R1 Oryza sativa subsp. japonica (Rice) OSJNBa0001K12.13Q8W5N7 Oryza sativa subsp. japonica (Rice) OsJ_31873 B9G683 Oryza sativasubsp. japonica (Rice) OsJ_31875 B9G684 Oryza sativa subsp. japonica(Rice) OSJNBa0001K12.3 Q8W5P5 Arabidopsis lyrata subsp. lyrata (Lyre-ARALYDRAFT_470376 D7KDA3 leaved rock-cress) Arabidopsis lyrata subsp.lyrata (Lyre- ARALYDRAFT_479855 D7L3B6 leaved rock-cress) Arabidopsislyrata subsp. lyrata (Lyre- ARALYDRAFT_491906 D7MDA9 leaved rock-cress)Arabidopsis lyrata subsp. lyrata (Lyre- ARALYDRAFT_914728 D7MGS9 leavedrock-cress)

Acetyl Transferase

The present disclosure describes enzymes that convert alcohols to fattyacetates.

In some embodiments, an acetyl transferase is used to catalyze theconversion of a fatty alcohol to a fatty acetate. An acetyl transferaseis an enzyme that has the ability to produce an acetate ester bytransferring the acetyl group from acetyl-CoA to an alcohol. In someembodiments, the acetyl transferase may have an EC number of 2.3.1.84.

The acetyl transferase, or the nucleic acid sequence that encodes it,can be isolated from various organisms, including but not limited to,organisms of the species Saccharomyces cerevisiae, Danaus plexippus,Heliotis virescens, Bombyx mori, Agrotis ipsilon, Agrotis segetum,Euonymus alatus. In exemplary embodiments, the acetyl transferasecomprises a sequence selected from GenBank Accession Nos. AY242066,AY242065, AY242064, AY242063, AY242062, EHJ65205, ACX53812,NP_001182381, EHJ65977, EHJ68573, KJ579226, GU594061. Additionalexemplary acetyl transferase peptides may be found in US2010/0199548,which is herein incorporated by reference.

Fatty acyl-ACP Thioesterase

Acyl-ACP thioesterase releases free fatty acids from Acyl-ACPs,synthesized from de novo fatty acid biosynthesis. The reactionterminates fatty acid biosynthesis. In plants, fatty acid biosynthesisoccurs in the plastid and thus requires plastid-localized acyl-ACPthioesterases. The main products of acyl-ACP thioesterase are oleate(C18:0) and to a lesser extent palmitate (C16:0) in the vegetativetissues of all plants. The released free fatty acids are re-esterifiedto coenzyme A in the plastid envelope and exported out of plastid.

There are two isoforms of acyl-ACP thioesterase, FatA and FatB.Substrate specificity of these isoforms determines the chain length andlevel of saturated fatty acids in plants. The highest activity of FatAis with C18:1-ACP. FatA has very low activities towards other acyl-ACPswhen compared with C18:1-ACP. FatB has highest activity with C16:0-ACP.It also has significant high activity with C18:1-ACP, followed byC18:0-ACP and C16:1-ACP. Kinetics studies of FatA and FatB indicate thattheir substrate specificities with different acyl-ACPs came from theKcat values, rather than from Km. Km values of the two isoforms withdifferent substrates are similar, in the micromolar order. Domainswapping of FatA and FatB indicates the N-terminus of the isoformsdetermines their substrate specificities (Salas J J and Ohlrogge J B(2002) Characterization of substrate specificity of plant FatA and FatBacyl-ACP thioesterases. Arch Biochem Biophys 403(1): 25-34). For thoseplants which predominantly accumulate medium-chain length saturatedfatty acids in seeds, they evolved with specialized FatB and/or FatAthioesterases (Voelker T and Kinney A J (2001) Variations in thebiosynthesis of seed-storage lipids. Annu Rev Plant Physiol Plant MolBiol 52: 335-361). For example, laurate (12:0) is the predominant seedoil in coconut. Correspondingly, the medium-chain specific acyl-ACPthioesterase activity was detected in coconut seeds.

In one embodiment, one or more fatty acyl-ACP thioesterases are selectedfrom the group consisting of Q41635, Q39473, P05521.2, AEM72519,AEM72520, AEM72521, AEM72523, AAC49784, CAB60830, EER87824, EER96252,ABN54268, AA077182, CAH09236, ACL08376, and homologs thereof.

Expression of Toxic Proteins or Polypeptides

The present disclosure describes a toxic protein, peptide, or smallmolecule that can be encoded by a recombinant microorganism. In someembodiments, the toxic protein, peptide, or small molecule isbiosynthetically produced along with an insect pheromone.

In some embodiments, the recombinant microorganism expresses one or morenucleic acid molecules encoding a protein or polypeptide which is toxicto an insect. In some embodiments, the toxic protein or polypeptide isfrom an entomopathogenic organism. In some embodiments, theentomopathogenic organism is selected from Bacillus thuringiensis,Pseudomonas aeruginosa, and Serratia marcescens. In a particularembodiment, the nucleic acid molecule encodes a Bacillus thuringiensistoxin.

In some embodiments, a recombinant microorganism is engineered toexpress a metabolic pathway which, when expressed, produces a smallmolecule that is toxic to an insect.

In exemplary embodiments, an insect pheromone produced by a recombinantmicroorganism described herein may be used to attract a pest insect, andsubsequently, the pest insect is eradicated with a toxic substance, suchas a toxic protein, peptide, or small molecule, which has beenco-produced by a recombinant microorganism described herein.

Biosynthesis of Pheromones Using a Recombinant Microorganism

As discussed above, in a first aspect, the present disclosure relates toa recombinant microorganism capable of producing a mono- orpoly-unsaturated C₆-C₂₄ fatty alcohol from an endogenous or exogenoussource of saturated C₆-C₂₄ fatty acyl-CoA. An illustrative embodiment ofthe first aspect is shown in FIG. 1. The blue lines designatebiochemical pathways used to produce a saturated acyl-CoA, which acts asa substrate for unsaturated fatty-acyl CoA conversion. The substrate tounsaturated fatty acyl-CoA conversion can be performed by endogenous orexogenous enzymes in a host. Green lines indicate conversions catalyzedby an exogenous nucleic acid molecule encoding for an enzyme.Accordingly, in some embodiments, the conversion of a saturated fattyacyl-CoA to a mono- or poly-unsaturated fatty acyl-CoA is catalyzed byat least one desaturase, which is encoded by an exogenous nucleic acidmolecule. In further embodiments, the conversion of the mono- orpoly-unsaturated fatty acyl-CoA to a mono- or poly-unsaturated fattyalcohol is catalyzed by at least one reductase, which is encoded by anexogenous nucleic acid molecule. The dashed grey lines indicatedownstream steps for the synthesis of pheromones, fragrances, flavors,and polymer intermediates, such as using an alcohol oxidase or oxidantto produce a mono- or poly-unsaturated fatty aldehyde, and an acetyltransferase or a chemical such as acetylchloride to produce a mono- orpoly-unsaturated fatty acetate. The red crosses indicate deleted or downregulated pathways native to the host, which increase flux towards theengineered pathway.

Accordingly, in one embodiment, the recombinant microorganism expresses:(a) at least one exogenous nucleic acid molecule encoding a fatty acyldesaturase that catalyzes the conversion of a saturated C₆-C₂₄ fattyacyl-CoA to a corresponding mono- or poly-unsaturated C₆-C₂₄ fattyacyl-CoA; and (b) at least one exogenous nucleic acid molecule encodinga fatty alcohol forming fatty-acyl reductase that catalyzes theconversion of the mono- or poly-unsaturated C₆-C₂₄ fatty acyl-CoA from(a) into the corresponding mono- or poly-unsaturated C₆-C₂₄ fattyalcohol. In some embodiments, the saturated C₆-C₂₄ fatty acyl-CoA can beproduced using endogenous enzymes in the host microorganism. In otherembodiments, the saturated C₆-C₂₄ fatty acyl-CoA can be produced usingone or more exogenous enzymes in the host microorganism.

As described above, a fatty acyl desaturase catalyzes the desaturationof the hydrocarbon chain on, e.g., a saturated fatty acyl-CoA moleculeto generate a corresponding unsaturated fatty acyl CoA molecule. In someembodiments, an exogenous fatty acyl desaturase can be selected andexpressed in a recombinant microorganism to catalyze the formation of atleast one double bond in fatty acyl-CoA molecule having from κ to 24carbons in the hydrocarbon chain. Accordingly, in some embodiments, thefatty-acyl desaturase is a desaturase capable of utilizing a fattyacyl-CoA as a substrate that has a chain length of 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 carbon atoms.

An exogenous fatty acyl desaturase described herein can be selected tocatalyze the desaturation at a desired position on the hydrocarbonchain. Accordingly, in some embodiments, the fatty-acyl desaturase iscapable of generating a double bond at position C5, C6, C7, C8, C9, C10,C11, C12, or C13, in the fatty acid or its derivatives, such as, forexample, fatty acid CoA esters.

One or more than one fatty acyl-CoA desaturase can be expressed in thehost to catalyze desaturation at multiple positions on the hydrocarbonchain. In some embodiments, the fatty acyl-CoA desaturase isheterologous to the host microorganism. Accordingly, various embodimentsprovide for recombinant microorganism comprised of at least oneexogenous nucleic acid molecule, which encodes a fatty acyl desaturasethat catalyzes the conversion of a saturated C₆-C₂₄ fatty acyl-CoA to acorresponding mono- or poly-unsaturated C₆-C₂₄ fatty acyl-CoA.

In one exemplary embodiment, the fatty-acyl desaturase is a Z11desaturase. The Z11 fatty-acyl desaturase catalyze double bond formationbetween the 11^(th) and 12^(th) carbons in the substrate relative to thecarbonyl group. In various embodiments described herein, the Z11desaturase, or the nucleic acid sequence that encodes it, can beisolated from organisms of the species Agrotis segetum, Amyeloistransitella, Argyrotaenia velutiana, Choristoneura rosaceana, Lamproniacapitella, Trichoplusia ni, Helicoverpa zea, or Thalassiosirapseudonana. Further Z11-desaturases, or the nucleic acid sequencesencoding them, can be isolated from Bombyx mori, Manduca sexta, Diatraeagrandiosella, Earias insulana, Earias vittella, Plutella xylostella,Bombyx mori or Diaphania nitidalis. In exemplary embodiments, the Z11desaturase comprises a sequence selected from GenBank Accession Nos.JX679209, JX964774, AF416738, AF545481, EU152335, AAD03775, AAF81787(SEQ ID NO: 39), and AY493438. In some embodiments, a nucleic acidsequence encoding a Z11 desaturase from organisms of the species Agrotissegetum, Amyelois transitella, Argyrotaenia velutiana, Choristoneurarosaceana, Lampronia capitella, Trichoplusia ni, Helicoverpa zea, orThalassiosira pseudonana is codon optimized. In some embodiments, theZ11 desaturase comprises a sequence selected from SEQ ID NOs: 9, 18, 24and 26 from Trichoplusia ni. In other embodiments, the Z11 desaturasecomprises a sequence selected from SEQ ID NOs: 10 and 16 from Agrotissegetum. In some embodiments, the Z11 desaturase comprises a sequenceselected from SEQ ID NOs: 11 and 23 from Thalassiosira pseudonana. Incertain embodiments, the Z11 desaturase comprises a sequence selectedfrom SEQ ID NOs: 12, 17 and 30 from Amyelois transitella. In furtherembodiments, the Z11 desaturase comprises a sequence selected from SEQID NOs: 13, 19, 25, 27 and 31 from Helicoverpa zea. In some embodiments,the Z11 desaturase comprises a chimeric polypeptide. In someembodiments, a complete or partial Z11 desaturase is fused to anotherpolypeptide. In certain embodiments, the N-terminal native leadersequence of a Z11 desaturase is replaced by an oleosin leader sequencefrom another species. In certain embodiments, the Z11 desaturasecomprises a sequence selected from SEQ ID NOs: 15, 28 and 29.

In certain embodiments, the Z11 desaturase catalyzes the conversion of afatty acyl-CoA into a mono- or poly-unsaturated product selected fromZ11-13:Acyl-CoA, E11-13:Acyl-CoA, (Z,Z)-7,11-13:Acyl-CoA,Z11-14:Acyl-CoA, E11-14:Acyl-CoA, (E,E)-9,11-14:Acyl-CoA,(E,Z)-9,11-14:Acyl-CoA, (Z,E)-9,11-14:Acyl-CoA, (Z,Z)-9,11-14:Acyl-CoA,(E,Z)-9,11-15:Acyl-CoA, (Z,Z)-9,11-15:Acyl-CoA, Z11-16:Acyl-CoA,E11-16:Acyl-CoA, (E,Z)-6,11-16:Acyl-CoA, (E,Z)-7,11-16:Acyl-CoA,(E,Z)-8,11-16:Acyl-CoA, (E,E)-9,11-16:Acyl-CoA, (E,Z)-9,11-16:Acyl-CoA,(Z,E)-9,11-16:Acyl-CoA, (Z,Z)-9,11-16:Acyl-CoA, (E,E)-11,13-16:Acyl-CoA,(E,Z)-11,13-16:Acyl-CoA, (Z,E)-11,13-16:Acyl-CoA,(Z,Z)-11,13-16:Acyl-CoA, (Z,E)-11,14-16:Acyl-CoA,(E,E,Z)-4,6,11-16:Acyl-CoA, (Z,Z,E)-7,11,13-16:Acyl-CoA,(E,E,Z,Z)-4,6,11,13-16:Acyl-CoA, Z11-17:Acyl-CoA,(Z,Z)-8,11-17:Acyl-CoA, Z11-18:Acyl-CoA, E11-18:Acyl-CoA,(Z,Z)-11,13-18:Acyl-CoA, (E,E)-11,14-18:Acyl-CoA, or combinationsthereof.

In another exemplary embodiment, the fatty-acyl desaturase is a Z9desaturase. The Z9 fatty-acyl desaturase catalyze double bond formationbetween the 9^(th) and 10^(th) carbons in the substrate relative to thecarbonyl group. In various embodiments described herein, the Z9desaturase, or the nucleic acid sequence that encodes it, can beisolated from organisms of the species Ostrinia furnacalis, Ostrinianobilalis, Choristoneura rosaceana, Lampronia capitella, Helicoverpaassulta, or Helicoverpa zea. In exemplary embodiments, the Z9 desaturasecomprises a sequence selected from GenBank Accession Nos. AY057862,AF243047, AF518017, EU152332, AF482906, and AAF81788. In someembodiments, a nucleic acid sequence encoding a Z9 desaturase is codonoptimized. In some embodiments, the Z9 desaturase comprises a sequenceset forth in SEQ ID NO: 20 from Ostrinia furnacalis. In otherembodiments, the Z9 desaturase comprises a sequence set forth in SEQ IDNO: 21 from Lampronia capitella. In some embodiments, the Z9 desaturasecomprises a sequence set forth in SEQ ID NO: 22 from Helicoverpa zea.

In certain embodiments, the Z9 desaturase catalyzes the conversion of afatty acyl-CoA into a monounsaturated or polyunsaturated productselected from Z9-11:Acyl-CoA, Z9-12:Acyl-CoA, E9-12:Acyl-CoA,(E,E)-7,9-12:Acyl-CoA, (E,Z)-7,9-12:Acyl-CoA, (Z,E)-7,9-12:Acyl-CoA,(Z,Z)-7,9-12:Acyl-CoA, Z9-13:Acyl-CoA, E9-13:Acyl-CoA,(E,Z)-5,9-13:Acyl-CoA, (Z,E)-5,9-13:Acyl-CoA, (Z,Z)-5,9-13:Acyl-CoA,Z9-14:Acyl-CoA, E9-14:Acyl-CoA, (E,Z)-4,9-14:Acyl-CoA,(E,E)-9,11-14:Acyl-CoA, (E,Z)-9,11-14:Acyl-CoA, (Z,E)-9,11-14:Acyl-CoA,(Z,Z)-9,11-14:Acyl-CoA, (E,E)-9,12-14:Acyl-CoA, (Z,E)-9,12-14:Acyl-CoA,(Z,Z)-9,12-14:Acyl-CoA, Z9-15:Acyl-CoA, E9-15:Acyl-CoA,(Z,Z)-6,9-15:Acyl-CoA, Z9-16:Acyl-CoA, E9-16:Acyl-CoA,(E,E)-9,11-16:Acyl-CoA, (E,Z)-9,11-16:Acyl-CoA, (Z,E)-9,11-16:Acyl-CoA,(Z,Z)-9,11-16:Acyl-CoA, Z9-17:Acyl-CoA, E9-18:Acyl-CoA, Z9-18:Acyl-CoA,(E,E)-5,9-18:Acyl-CoA, (E,E)-9,12-18:Acyl-CoA, (Z,Z)-9,12-18:Acyl-CoA,(Z,Z,Z)-3,6,9-18:Acyl-CoA, (E,E,E)-9,12,15-18:Acyl-CoA,(Z,Z,Z)-9,12,15-18:Acyl-CoA, or combinations thereof.

Desaturation of a saturated C₆-C₂₄ fatty acyl-CoA can proceed through aplurality of reactions to produce a poly-unsaturated C₆-C₂₄ fattyacyl-CoA. In some embodiments, the recombinant microorganism may expressa bifunctional desaturase capable of catalyzing the formation at leasttwo double bonds. In some embodiments, the recombinant microorganism mayexpress more than one exogenous nucleic acid molecule encoding more thanone fatty-acyl desaturase that catalyzes the conversion of a saturatedC₆-C₂₄ fatty acyl-CoA to a corresponding poly-unsaturated C₆-C₂₄ fattyacyl-CoA. For example, the recombinant microorganism may express anexogenous nucleic acid molecule encoding a Z11 desaturase and anotherexogenous nucleic acid molecule encoding a Z9 desaturase. Thus, theresultant poly-unsaturated fatty acyl-CoA would have a double bondbetween the 9^(th) and 10^(th) carbon and another double bond betweenthe 11^(th) and 12^(th) carbon.

In some embodiments, the recombinant microorganism may express afatty-acyl conjugase that acts independently or together with afatty-acyl desaturase to catalyze the conversion of a saturated ormonounsaturated fatty acyl-CoA to a conjugated polyunsaturated fattyacyl-CoA.

In one embodiment, the disclosure provides a recombinant microorganismcapable of producing a polyunsaturated C₆-C₂₄ fatty alcohol from anendogenous or exogenous source of saturated or monounsaturated C₆-C₂₄fatty acyl-CoA, wherein the recombinant microorganism expresses: (a) atleast one exogenous nucleic acid molecule encoding a fatty acylconjugase that catalyzes the conversion of a saturated ormonounsaturated C₆-C₂₄ fatty acyl-CoA to a corresponding polyunsaturatedC₆-C₂₄ fatty acyl-CoA; and (b) at least one exogenous nucleic acidmolecule encoding a fatty alcohol forming fatty-acyl reductase thatcatalyzes the conversion of the polyunsaturated C₆-C₂₄ fatty acyl-CoAfrom (a) into the corresponding polyunsaturated C₆-C₂₄ fatty alcohol.

In another embodiment, the recombinant microorganism expresses at leasttwo exogenous nucleic acid molecules encoding fatty-acyl conjugases thatcatalyze the conversion of a saturated or monounsaturated C₆-C₂₄ fattyacyl-CoA to a corresponding polyunsaturated C₆-C₂₄ fatty acyl-CoA.

In a further embodiment, the disclosure provides a recombinantmicroorganism capable of producing a polyunsaturated C₆-C₂₄ fattyalcohol from an endogenous or exogenous source of saturated ormonounsaturated C₆-C₂₄ fatty acyl-CoA, wherein the recombinantmicroorganism expresses: (a) at least one exogenous nucleic acidmolecule encoding a fatty-acyl desaturase and at least one exogenousnucleic acid molecule encoding a fatty acyl conjugase that catalyze theconversion of a saturated or monounsaturated C₆-C₂₄ fatty acyl-CoA to acorresponding polyunsaturated C₆-C₂₄ fatty acyl-CoA; and (b) at leastone exogenous nucleic acid molecule encoding a fatty alcohol formingfatty-acyl reductase that catalyzes the conversion of thepolyunsaturated C₆-C₂₄ fatty acyl-CoA from (a) into the correspondingpolyunsaturated C₆-C₂₄ fatty alcohol.

In another embodiment, the recombinant microorganism expresses at leasttwo exogenous nucleic acid molecules encoding fatty-acyl desaturases andat least two exogenous nucleic acid molecules encoding fatty-acylconjugases that catalyze the conversion of a saturated ormonounsaturated C₆-C₂₄ fatty acyl-CoA to a corresponding polyunsaturatedC₆-C₂₄ fatty acyl-CoA.

In yet a further embodiment, the fatty-acyl conjugase is a conjugasecapable of utilizing a fatty acyl-CoA as a substrate that has a chainlength of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, or 24 carbon atoms.

In certain embodiments, the conjugase, or the nucleic acid sequence thatencodes it, can be isolated from organisms of the species Cydiapomonella, Cydia nigricana, Lobesia botrana, Myelois cribrella, Plodiainterpunctella, Dendrolimus punctatus, Lampronia capitella, Spodopteralitura, Amyelois transitella, Manduca sexta, Bombyx mori, Calendulaofficinalis, Trichosanthes kirilowii, Punica granatum, Momordicacharantia, Impatiens balsamina, and Epiphyas postvittana. In exemplaryembodiments, the conjugase comprises a sequence selected from GenBankAccession No. or Uniprot database: A0A059TBF5, A0A0M3L9E8, A0A0M3L9S4,A0A0M3LAH8, A0A0M3LAS8, A0A0M3LAH8, B6CBS4, XP_013183656.1,XP_004923568.2, ALA65425.1, NP_001296494.1, NP_001274330.1, Q4A181,Q75PL7, Q9FPP8, AY178444, AY178446, AF182521, AF182520, Q95UJ3.

As described above, a fatty acyl reductase catalyzes the reduction of acarbonyl group, e.g., on an unsaturated fatty acyl-CoA molecule togenerate a corresponding unsaturated fatty acid molecule. In someembodiments, the fatty alcohol forming fatty acyl CoA reductase isheterologous to the microorganism. Accordingly, various embodimentsprovide for recombinant microorganism comprised of at least oneexogenous nucleic acid molecule, which encodes a fatty alcohol formingfatty acyl reductase that catalyzes the reduction of a carbonyl group onan unsaturated fatty acyl-CoA molecule to generate a correspondingunsaturated fatty acid molecule.

In some embodiments, the fatty acyl reductase is from an organism of thespecies Agrotis segetum, Spodoptera littoralis, or Helicoverpa amigera.In some embodiments, a nucleic acid sequence encoding a fatty-acylreductase is codon optimized. In some embodiments, the fatty acylreductase comprises a sequence set forth in SEQ ID NO: 1 from Agrotissegetum. In other embodiments, the fatty acyl reductase comprises asequence set forth in SEQ ID NO: 2 from Spodoptera littoralis. In someembodiments, the fatty acyl reductase comprises a sequence selected fromSEQ ID NOs: 3 and 32 from Helicoverpa armigera.

In exemplary embodiments, the fatty-acyl reductase catalyzes theconversion of a mono- or poly-unsaturated fatty acyl-CoA into a fattyalcohol product selected from (Z)-3-hexenol, (Z)-3-nonenol,(Z)-5-decenol, (E)-5-decenol, (Z)-7-dodecenol, (E)-8-dodecenol,(Z)-8-dodecenol, (Z)-9-dodecenol, (Z)-9-tetradecenol, (Z)-9-hexadecenol,(Z)-11-tetradecenol, (Z)-7-hexadecenol, (Z)-11-hexadecenol,(E)-11-tetradecenol, or (Z,Z)-11,13-hexadecadienol,(11Z,13E)-hexadecadienol, (E,E)-8,10-dodecadienol,(E,Z)-7,9-dodecadienol, (Z)-13-octadecenol, or combinations thereof.

In some embodiments, a recombinant microorganism described herein caninclude a plurality of fatty acyl reductases. Accordingly, in suchembodiments, the recombinant microorganism expresses at least twoexogenous nucleic acid molecules, which encode fatty-acyl reductasesthat catalyze the conversion of the mono- or poly-unsaturated C₆-C₂₄fatty acyl-CoA into the corresponding mono- or poly-unsaturated C₆-C₂₄fatty alcohol.

As discussed above, in a second aspect, the application relates to arecombinant microorganism capable of producing an unsaturated C₆-C₂₄fatty alcohol from an endogenous or exogenous source of C₆-C₂₄ fattyacid. An illustrative embodiment of the second aspect is shown in FIG.2. The blue lines designate biochemical pathways endogenous to the host,e.g., pathways for converting an n-alkane, fatty alcohol, or fattyaldehyde to a fatty acid, or the conversion of a fatty acid tofatty-acyl-CoA, acetyl-CoA, or dicarboxylic acid. The substrate tounsaturated fatty acid conversion can be performed by endogenous orexogenous enzymes in a host. Yellow lines indicate conversions catalyzedby an exogenous nucleic acid molecule encoding for an enzyme.Accordingly, in some embodiments, the conversion of a saturated fattyacid to a saturated fatty acyl-ACP can be catalyzed by at least onesaturated fatty acyl-ACP synthetase, wherein the fatty acyl-ACPsynthetase is encoded by an exogenous nucleic acid molecule. In furtherembodiments, the conversion of the saturated fatty acyl-ACP to a mono-or poly-unsaturated fatty acyl-ACP can be catalyzed by at least onefatty acyl-ACP desaturase, wherein the fatty acyl-ACP desaturase isencoded by an exogenous nucleic acid molecule. In still furtherembodiments, the mono- or poly-unsaturated fatty acyl-ACP can beelongated by at least 2 carbons relative using a fatty acid synthasecomplex and a carbon source, e.g., malonyl-ACP. In one such embodiment,the conversion of the mono- or poly-unsaturated fatty acyl-ACP to acorresponding two carbon elongated mono- or poly-unsaturated fattyacyl-ACP can be catalyzed by at least one fatty acid synthase complex,wherein the fatty acid synthase complex is encoded by one or moreexogenous nucleic acid molecules. In yet further embodiments, theconversion of the elongated mono- or poly-unsaturated fatty acyl-ACP toa mono- or poly-unsaturated fatty aldehyde can be catalyzed by a fattyaldehyde forming fatty acyl reductase, wherein the fatty aldehydeforming fatty acyl reductase is encoded by an exogenous nucleic acidmolecule. In some embodiments, the mono- or poly-unsaturated fattyaldehyde can be converted to a corresponding mono- or poly-unsaturatedfatty alcohol, wherein the substrate to product conversion is catalyzedby a dehydrogenase, wherein the dehydrogenase is encoded by anendogenous or exogenous nucleic acid molecule. The dashed lines indicatedownstream steps of the disclosure, such as utilizing an acetyltransferase or metathesis, or subsequent chemical transformations toproduce functionalized pheromones. The red crosses indicate deleted ordown regulated pathways native to the host, which increase flux towardsthe engineered pathway.

In one embodiment, the recombinant microorganism expresses (a): at leastone exogenous nucleic acid molecule encoding an acyl-ACP synthetase thatcatalyzes the conversion of a C₆-C₂₄ fatty acid to a correspondingsaturated C₆-C₂₄ fatty acyl-ACP; (b) at least one exogenous nucleic acidmolecule encoding a fatty-acyl-ACP desaturase that catalyzes theconversion of a saturated C₆-C₂₄ fatty acyl-ACP to a corresponding mono-or poly-unsaturated C₆-C₂₄ fatty acyl-ACP; (c) one or more endogenous orexogenous nucleic acid molecules encoding a fatty acid synthase complexthat catalyzes the conversion of the mono- or poly-unsaturated C₆-C₂₄fatty acyl-ACP from (b) to a corresponding mono- or poly-unsaturatedC₆-C₂₄ fatty acyl-ACP with a two carbon elongation relative to theproduct of (b); (d): at least one exogenous nucleic acid moleculeencoding a fatty aldehyde forming fatty-acyl reductase that catalyzesthe conversion of the mono- or poly-unsaturated C₆-C₂₄ fatty acyl-ACPfrom (c) into a corresponding mono- or poly-unsaturated C₆-C₂₄ fattyaldehyde; and (e) at least one endogenous or exogenous nucleic acidmolecule encoding a dehydrogenase that catalyzes the conversion of themono- or poly-unsaturated C₆-C₂₄ fatty aldehyde C₆-C₂₄ from (d) into acorresponding mono- or poly-unsaturated C₆-C₂₄ fatty alcohol. In someembodiments, the C₆-C₂₄ fatty acid can be produced using endogenousenzymes in the host microorganism. In other embodiments, the saturatedC₆-C₂₄ fatty acid can be produced by one or more exogenous enzymes inthe host microorganism.

In some embodiments, the recombinant microorganism disclosed hereinincludes an acyl-ACP synthetase to catalyze the conversion of a C₆-C₂₄fatty acid to a corresponding saturated C₆-C₂₄ fatty acyl-ACP. In someembodiments the acyl-ACP synthetase is a synthetase capable of utilizinga fatty acid as a substrate that has a chain length of 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 carbon atoms.In exemplary embodiments, the recombinant microorganism can include aheterologous the acyl-ACP synthetase from an organism of the speciesVibrio harveyi, Rhodotorula glutinis, or Yarrowia lipolytica.

In some embodiments, the recombinant microorganism includes a fattyacyl-ACP desaturase. In some embodiments, the fatty acyl-ACP desaturaseis a soluble desaturase. In other embodiments, the fatty-acyl-ACPdesaturase is from an organism of the species Pelargonium hortorum,Asclepias syriaca, or Uncaria tomentosa.

In some embodiments, the recombinant microorganism includes a fatty acidsynthase complex. In some embodiments, the one or more nucleic acidmolecules encoding the fatty acid synthase complex are endogenousnucleic acid molecules. In other embodiments, the one or more nucleicacid molecules encoding a fatty acid synthase complex are exogenousnucleic acid molecules.

In some embodiments, the recombinant microorganism disclosed hereinincludes a fatty aldehyde forming fatty-acyl reductase which catalyzesthe conversion of a C₆-C₂₄ fatty acyl-ACP to the corresponding C₆-C₂₄fatty aldehyde. In exemplary embodiments, the fatty aldehyde formingfatty-acyl reductase is from an organism of the species Pelargoniumhortorum, Asclepias syriaca, and Uncaria tomentosa. In some embodiments,the recombinant microorganism includes a dehydrogenase to convert theunsaturated fatty aldehyde to a corresponding unsaturated fatty alcohol.In some embodiments, the nucleic acid molecule encoding thedehydrogenase is endogenous to the recombinant microorganism. In otherembodiments, the nucleic acid molecule encoding a dehydrogenase isexogenous to the recombinant microorganism. In exemplary embodiments,the endogenous or exogenous nucleic acid molecule encoding adehydrogenase is isolated from organisms of the species Saccharomycescerevisiae, Escherichia coli, Yarrowia lipolytica, or Candidatropicalis.

As discussed above, in a third aspect, the application relates to arecombinant microorganism capable of producing an unsaturated C₆-C₂₄fatty alcohol from an endogenous or exogenous source of C₆-C₂₄ fattyacid. An illustrative embodiment of the second aspect is shown in FIG.3. The blue lines designate biochemical pathways endogenous to the host,e.g., pathways for converting an n-alkane, fatty alcohol, or fattyaldehyde to a fatty acid, or the conversion of a fatty acid tofatty-acyl-CoA, acetyl-CoA, or dicarboxylic acid. The substrate tounsaturated fatty acid conversion can be performed by endogenous orexogenous enzymes in a host. Yellow lines indicate conversions catalyzedby an exogenous nucleic acid molecule encoding for an enzyme.Accordingly, in some embodiments, the conversion of a saturated fattyacid to a saturated fatty acyl-ACP can be catalyzed by at least onesaturated fatty acyl-ACP synthetase, wherein the fatty acyl-ACPsynthetase is encoded by an exogenous nucleic acid molecule. Thenon-native saturated fatty acyl-ACP thioesters create a substratesuitable for desaturation and distinct from CoA-thioesters used forbeta-oxidation or fatty acid elongation. In further embodiments, theconversion of the saturated fatty acyl-ACP to a mono- orpoly-unsaturated fatty acyl-ACP can be catalyzed by at least one fattyacyl-ACP desaturase, wherein the fatty acyl-ACP desaturase is encoded byan exogenous nucleic acid molecule. In still further embodiments, themono- or poly-unsaturated fatty acyl-ACP can be converted to acorresponding mono- or poly-unsaturated fatty acid by a fatty-acyl-ACPthioesterase. In a particular embodiment, soluble fatty acyl-ACPthioesterases can be used to release free fatty acids for reactivationto a CoA thioester. Fatty acyl-ACP thioesterases including Q41635,Q39473, P05521.2, AEM72519, AEM72520, AEM72521, AEM72523, AAC49784,CAB60830, EER87824, EER96252, ABN54268, AA077182, CAH09236, ACL08376,and homologs thereof may be used. In an additional embodiment, the mono-or poly-unsaturated fatty acyl-CoA can be elongated by at least 2carbons relative using an elongase and a carbon source, e.g.,malonyl-ACP. In yet further embodiments, the conversion of the elongatedmono- or poly-unsaturated fatty acyl-CoA to a mono- or poly-unsaturatedfatty alcohol can be catalyzed by a fatty alcohol forming fatty acylreductase, wherein the fatty alcohol forming fatty acyl reductase isencoded by an exogenous nucleic acid molecule. The dashed lines indicatedownstream steps of the disclosure, such as utilizing an acetyltransferase or metathesis, or subsequent chemical transformations toproduce functionalized pheromones. The red crosses indicate deleted ordown regulated pathways native to the host, which increase flux towardsthe engineered pathway.

The fatty alcohols produced as taught herein can be further converted toproduce downstream products such as insect pheromones, fragrances,flavors, and polymer intermediates, which utilize aldehydes or acetatefunctional groups. Thus, in some embodiments, the recombinantmicroorganism further comprises at least one endogenous or exogenousnucleic acid molecule encoding an alcohol oxidase or an alcoholdehydrogenase, wherein the alcohol oxidase or alcohol dehydrogenase iscapable of catalyzing the conversion of a C₆-C₂₄ fatty alcohol into acorresponding C₆-C₂₄ fatty aldehyde. In other embodiments, therecombinant microorganism can further comprise at least one endogenousor exogenous nucleic acid molecule encoding an acetyl transferasecapable of catalyzing the conversion of a C₆-C₂₄ fatty alcohol into acorresponding C₆-C₂₄ fatty acetate. In certain embodiments, the acetyltransferase, or the nucleic acid sequence that encodes it, can beisolated from organisms of the species Saccharomyces cerevisiae, Danausplexippus, Heliotis virescens, Bombyx mori, Agrotis Ipsilon, Agrotissegetum, Euonymus alatus. In exemplary embodiments, the acetyltransferase comprises a sequence selected from GenBank Accession Nos.AY242066, AY242065, AY242064, AY242063, AY242062, EHJ65205, ACX53812,NP_001182381, EHJ65977, EHJ68573, KJ579226, GU594061.

Recombinant Microorganism

The disclosure provides microorganisms that can be engineered to expressvarious exogenous enzymes.

In various embodiments described herein, the recombinant microorganismis a eukaryotic microorganism. In some embodiments, the eukaryoticmicroorganism is a yeast. In exemplary embodiments, the yeast is amember of a genus selected from the group consisting of Yarrowia,Candida, Saccharomyces, Pichia, Hansenula, Kluyveromyces, Issatchenkia,Zygosaccharomyces, Debaryomyces, Schizosaccharomyces, Pachysolen,Cryptococcus, Trichosporon, Rhodotorula, and Myxozyma.

The present inventors have discovered that oleaginous yeast, such asCandida and Yarrowia, have a surprisingly high tolerance to the C₆-C₂₄fatty alcohol substrates and products. Accordingly, in one suchexemplary embodiment, the recombinant microorganism of the invention isan oleaginous yeast. In further embodiments, the oleaginous yeast is amember of a genus selected from the group consisting of Yarrowia,Candida, Rhodotorula, Rhodosporidium, Cryptococcus, Trichosporon, andLipomyces. In even further embodiments, the oleaginous yeast is a memberof a species selected from Yarrowia lipolytica, Candida tropicalis,Rhodosporidium toruloides, Lipomyces starkey, L. lipoferus, C. revkaufi,C. pulcherrima, C. utilis, Rhodotorula minuta, Trichosporon pullans, T.cutaneum, Cryptococcus curvatus, R. glutinis, and R. graminis.

In some embodiments, the recombinant microorganism is a prokaryoticmicroorganism. In exemplary embodiments, the prokaryotic microorganismis a member of a genus selected from the group consisting ofEscherichia, Clostridium, Zymomonas, Salmonella, Rhodococcus,Pseudomonas, Bacillus, Lactobacillus, Enterococcus, Alcaligenes,Klebsiella, Paenibacillus, Arthrobacter, Corynebacterium, andBrevibacterium.

In some embodiments, the recombinant microorganism is used to produce amono- or poly-unsaturated C₆-C₂₄ fatty alcohol, aldehyde, or acetatedisclosed herein.

Accordingly, in another aspect, the present inventions provide a methodof producing a mono- or poly-unsaturated C₆-C₂₄ fatty alcohol, aldehyde,or acetate using a recombinant microorganism described herein. In oneembodiment, the method comprises cultivating the recombinantmicroorganism in a culture medium containing a feedstock providing acarbon source until the mono- or poly-unsaturated C₆-C₂₄ fatty alcohol,aldehyde, or acetate is produced. In a further embodiment, the mono- orpoly-unsaturated C₆-C₂₄ fatty alcohol, aldehyde, or acetate isrecovered. Recovery can be by methods known in the art, such asdistillation, membrane-based separation gas stripping, solventextraction, and expanded bed adsorption.

In some embodiments, the feedstock comprises a carbon source. In variousembodiments described herein, the carbon source may be selected fromsugars, glycerol, alcohols, organic acids, alkanes, fatty acids,lignocellulose, proteins, carbon dioxide, and carbon monoxide. In afurther embodiment, the sugar is selected from the group consisting ofglucose, fructose, and sucrose.

Enzyme Engineering

The enzymes in the recombinant microorganism can be engineered toimprove one or more aspects of the substrate to product conversion.Non-limiting examples of enzymes that can be further engineered for usein methods of the disclosure include a desaturase (e.g., a fattyacyl-CoA desaturase or fatty acyl-ACP desaturase), an acyl-ACPsynthetase, a fatty acid synthetase, a fatty acid synthase complex, anacetyl transferase, dehydrogenase, and an alcohol oxidase, andcombinations thereof. These enzymes can be engineered for improvedcatalytic activity, improved selectivity, improved stability, improvedtolerance to various fermentations conditions (temperature, pH, etc.),or improved tolerance to various metabolic substrates, products,by-products, intermediates, etc.

Desaturase enzymes can be engineered for improved catalytic activity inthe desaturation of an unsaturated substrate, for improved hydrocarbonselectivity, for improved selectivity of a Z product over an E product,or an E product over a Z product. For example, the Z9 fatty-acyldesaturase can be engineered to improve the yield in the substrate toproduct conversion of a saturated fatty acyl-CoA to the correspondingunsaturated fatty acyl-CoA, and, in addition or in the alternative, toimprove selectivity of the desaturation at the 9 position to produce acorresponding Z-9 fatty acyl-CoA. In further non-limiting examples, thefatty acyl-ACP synthetase can be engineered for improved ACP ligationactivity; a fatty acid synthase complex enzyme can be engineered forimproved catalytic activity of elongation of a fatty acid substrate; afatty alcohol forming fatty acyl-reductase can be engineered forimproved catalytic activity in the reduction of a fatty acyl-CoA to acorresponding fatty alcohol; a fatty aldehyde forming fattyacyl-reductase can be engineered for improved catalytic activity in thereduction of a fatty acyl-ACP to a corresponding fatty aldehyde; adehydrogenase can be engineered for improved catalytic activity in theconversion of a fatty acyl-ACP to a corresponding fatty alcohol; analcohol oxidase can be engineered for improved catalytic activity in theconversion of a fatty alcohol into a corresponding fatty aldehyde; andan acetyl transferase can be engineered for improved catalytic activityin the conversion of a fatty alcohol into a corresponding fatty acetate.

The term “improved catalytic activity” as used herein with respect to aparticular enzymatic activity refers to a higher level of enzymaticactivity than that measured relative to a comparable non-engineeredenzyme, such as a non-engineered desaturase (e.g. fatty acyl-CoAdesaturase or fatty acyl-ACP desaturase), fatty alcohol or aldehydeforming fatty-acyl reductase, acyl-ACP synthetase, fatty acidsynthetase, fatty acid synthase complex, acyl transferase,dehydrogenase, or an alcohol oxidase enzyme. For example, overexpressionof a specific enzyme can lead to an increased level of activity in thecells for that enzyme. Mutations can be introduced into a desaturase(e.g. fatty acyl-CoA desaturase or fatty acyl-ACP desaturase), a fattyalcohol or aldehyde forming fatty-acyl reductase, a acyl-ACP synthetase,a fatty acid synthetase, a fatty acid synthase complex, a acyltransferase, a dehydrogenase, or an alcohol oxidase enzyme resulting inengineered enzymes with improved catalytic activity. Methods to increaseenzymatic activity are known to those skilled in the art. Suchtechniques can include increasing the expression of the enzyme byincreasing plasmid copy number and/or use of a stronger promoter and/oruse of activating riboswitches, introduction of mutations to relievenegative regulation of the enzyme, introduction of specific mutations toincrease specific activity and/or decrease the K_(M) for the substrate,or by directed evolution. See, e.g., Methods in Molecular Biology (vol.231), ed. Arnold and Georgiou, Humana Press (2003).

Metabolic Engineering—Enzyme Overexpression and GeneDeletion/Downregulation for Increased Pathway Flux

In various embodiments described herein, the exogenous and endogenousenzymes in the recombinant microorganism participating in thebiosynthesis pathways described herein may be overexpressed.

The terms “overexpressed” or “overexpression” refers to an elevatedlevel (e.g., aberrant level) of mRNAs encoding for a protein(s), and/orto elevated levels of protein(s) in cells as compared to similarcorresponding unmodified cells expressing basal levels of mRNAs orhaving basal levels of proteins. In particular embodiments, mRNA(s) orprotein(s) may be overexpressed by at least 2-fold, 3-fold, 4-fold,5-fold, 6-fold, 8-fold, 10-fold, 12-fold, 15-fold or more inmicroorganisms engineered to exhibit increased gene mRNA, protein,and/or activity.

In some embodiments, a recombinant microorganism of the disclosure isgenerated from a host that contains the enzymatic capability tosynthesize a substrate fatty acid. In this specific embodiment it can beuseful to increase the synthesis or accumulation of a fatty acid to, forexample, increase the amount of fatty acid available to an engineeredfatty alcohol production pathway.

In some embodiments, it may be useful to increase the expression ofendogenous or exogenous enzymes involved in the fatty alcohol, aldehyde,or acetate production pathway to increase flux from the fatty acid tothe fatty alcohol, aldehyde, or acetate, thereby resulting in increasedsynthesis or accumulation of the fatty alcohol, aldehyde, or acetate.

In some embodiments, it may be useful to increase the expression ofendogenous or exogenous enzymes to increase intracellular levels of acoenzyme. In one embodiment, the coenzyme is NADH. In anotherembodiment, the coenzyme is NADPH. In one embodiment, the expression ofproteins in the pentose phosphate pathway is increased to increase theintracellular levels of NADPH. The pentose phosphate pathway is animportant catabolic pathway for supplying reduction equivalents and animportant anabolic pathway for biosynthesis reactions. In oneembodiment, a glucose-6-phosphate dehydrogenase that convertsglucose-6-phosphate to 6-phospho D-glucono-1,5-lactone is overexpressed.In some embodiments, the glucose-6-phosphate dehydrogenase is ZWF1 fromyeast. In another embodiment, the glucose-6-phosphate dehydrogenase isZWF1 (YNL241C) from Saccharomyces cerevisiae. In one embodiment, aglucose-6-phosphate-1-dehydrogenase that convertsD-glucopyranose-6-phosphate to 6-phospho D-glucono-1,5-lactone isoverexpressed. In another embodiment, theglucose-6-phosphate-1-dehydrogenase is zwf from bacteria. In certainembodiments, the glucose-6-phosphate-1-dehydrogenase is zwf (NP_416366)from E. coli. In one embodiment, a 6-phosphogluconolactonase thatconverts 6-phospho D-glucono-1,5-lactone to D-gluconate 6-phosphate isoverexpressed. In some embodiments, the 6-phosphogluconolactonase isSOL3 of yeast. In certain embodiments, the 6-phosphogluconolactonase isSOL3 (NP_012033) of Saccharomyces cerevisiae. In some embodiments, the6-phosphogluconolactonase is SOL4 of yeast. In certain embodiments, the6-phosphogluconolactonase is SOL4 (NP_011764) of Saccharomycescerevisiae. In some embodiments, the 6-phosphogluconolactonase is pgl ofbacteria. In certain embodiments, the 6-phosphogluconolactonase is pgl(NP_415288) of E. coli. In one embodiment, a 6-phosphogluconatedehydrogenase that converts D-gluconate 6-phosphate to D-ribulose5-phosphate is overexpressed. In some embodiments, the6-phosphogluconate dehydrogenase is GND1 from yeast. In certainembodiments, the 6-phosphogluconate dehydrogenase is GND1 (YHR183W) fromSaccharomyces cerevisiae. In some embodiments, the 6-phosphogluconatedehydrogenase is GND2 from yeast. In certain embodiments, the6-phosphogluconate dehydrogenase is GND2 (YGR256W) from Saccharomycescerevisiae. In some embodiments, the 6-phosphogluconate dehydrogenase isgnd from bacteria. In certain embodiments, the 6-phosphogluconatedehydrogenase is gnd (NP_416533) from E. coli. In one embodiment, atransaldolase that interconverts D-glyceraldehyde 3-phosphate andD-sedoheptulose 7-phosphate to β-D-fructofuranose 6-phosphate andD-erythrose 4-phosphate is overexpressed. In some embodiments, thetransaldolase is TAL1 of yeast. In certain embodiments, thetransaldolase is TAL1 (NP_013458) of Saccharomyces cerevisiae. In someembodiments, the transaldolase is NQM1 of yeast. In certain embodiments,the transaldolase is NQM1 (NP_011557) of Saccharomyces cerevisiae. Insome embodiments, the transaldolase is tal of bacteria. In certainembodiments, the transaldolase is talB (NP_414549) of E. coli. Incertain embodiments, the transaldolase is talA (NP_416959) of E. coli.In one embodiment, a transketolase that interconverts D-erythrose4-phosphate and D-xylulose 5-phosphate to β-D-fructofuranose 6-phosphateand D-glyceraldehyde 3-phosphate and/or interconverts D-sedoheptulose7-phosphate and D-glyceraldehyde 3-phosphate to D-ribose 5-phosphate andD-xylulose 5-phosphate is overexpressed. In some embodiments, thetransketolase is TKL1 of yeast. In certain embodiments, thetransketolase is TKL1 (NP_015399) of Saccharomyces cerevisiae. In someembodiments, the transketolase is TKL2 of yeast. In some embodiments,the transketolase is TKL2 (NP_009675) of Saccharomyces cerevisiae. Insome embodiments, the transketolase is tkt of bacteria. In certainembodiments, the transketolase is tktA (YP 026188) of E. coli. Incertain embodiments, the transketolase is tktB (NP_416960) of E. coli.In one embodiment, a ribose-5-phosphate ketol-isomerase thatinterconverts D-ribose 5-phosphate and D-ribulose 5-phosphate isoverexpressed. In some embodiments, the ribose-5-phosphateketol-isomerase is RKI1 of yeast. In certain embodiments, theribose-5-phosphate ketol-isomerase is RKI1 (NP_014738) of Saccharomycescerevisiae. In some embodiments, the ribose-5-phosphate isomerase is rpiof bacteria. In certain embodiments, the ribose-5-phosphate isomerase isrpiA (NP_417389) of E. coli. In certain embodiments, theribose-5-phosphate isomerase is rpiB (NP_418514) of E. coli. In oneembodiment, a D-ribulose-5-phosphate 3-epimerase that interconvertsD-ribulose 5-phosphate and D-xylulose 5-phosphate is overexpressed. Insome embodiments, the D-ribulose-5-phosphate 3-epimerase is RPE1 ofyeast. In certain embodiments, the D-ribulose-5-phosphate 3-epimerase isRPE1 (NP_012414) of Saccharomyces cerevisiae. In some embodiments, theD-ribulose-5-phosphate 3-epimerase is rpe of bacteria. In certainembodiments, the D-ribulose-5-phosphate 3-epimerase is rpe (NP_417845)of E. coli.

In one embodiment, the expression of an NADP+-dependent isocitratedehydrogenase is increased to increase intracellular levels of acoenzyme. In one embodiment, an NADP+ dependent isocitrate dehydrogenaseoxidizes D-threo-isocitrate to 2-oxoglutarate with concomitantgeneration of NADPH. In another embodiment, an NADP+ dependentisocitrate dehydrogenase oxidizes D-threo-isocitrate to 2-oxalosuccinatewith concomitant generation of NADPH. In some embodiments, theNADP+-dependent isocitrate dehydrogenase is IDP from yeast. In certainembodiments, the NADP+-dependent isocitrate dehydrogenase is IDP2(YLR174W) from Saccharomyces cerevisiae. In some embodiments, theNADP+-dependent isocitrate dehydrogenase is icd from bacteria. Incertain embodiments, the NADP+-dependent isocitrate dehydrogenase is icd(NP_415654) from E. coli.

In some embodiments, the expression of a malic enzyme thatdecarboxylates malate to pyruvate with concomitant generation of NADH orNADPH is increased to increase intracellular levels of a coenzyme. Inone embodiment, the malic enzyme is NAD+ dependent. In anotherembodiment, the malic enzyme is NADP+ dependent. In one embodiment, themalic enzyme is an NAD+ dependent malate dehydrogenase from bacteria. Insome embodiments, the NAD+ dependent malate dehydrogenase is maeA(NP_415996) from E. coli. In some embodiments, the NAD+ dependent malatedehydrogenase is maeE (CAQ68119) from Lactobacillus casei. In anotherembodiment, the malic enzyme is a mitochondrial NAD+ dependent malatedehydrogenase from yeast. In some embodiments, the NAD+ dependent malatedehydrogenase is MAE1 (YKL029C) from S. cerevisiae. In anotherembodiment, the malic enzyme is a mitochondrial NAD+ dependent malatedehydrogenase from a parasitic nematode. In some embodiments, the NAD+dependent malate dehydrogenase is M81055 from Ascaris suum. In oneembodiment, the malic enzyme is an NADP+ dependent malate dehydrogenasefrom bacteria. In some embodiments, the NADP+ dependent malatedehydrogenase is maeB (NP_416958) from E. coli. In one embodiment, themalic enzyme is an NADP+ dependent malate dehydrogenase from corn. Insome embodiments, the NADP+ dependent malate dehydrogenase is mel fromZea mays.

In some embodiments, the expression of an aldehyde dehydrogenase thatoxidizes an aldehyde to a carboxylic acid with concomitant generation ofNADH or NADPH is increased to increase intracellular levels of acoenzyme. In one embodiment, the aldehyde dehydrogenase is NAD+dependent. In another embodiment, the aldehyde dehydrogenase is NADP+dependent. In one embodiment, the aldehyde dehydrogenase is an NAD+dependent aldehyde dehydrogenase from bacteria. In some embodiments, theNAD+ dependent aldehyde dehydrogenase is aldA (NP_415933) from E. coli.In another embodiment, the aldehyde dehydrogenase is a cytosolic NADP+dependent aldehyde dehydrogenase from yeast. In some embodiments, theNADP+ dependent aldehyde dehydrogenase is ALD6 (YPL061W) from S.cerevisiae. In another embodiment, the aldehyde dehydrogenase is acytosolic NADP+ dependent aldehyde dehydrogenase from bacteria. In someembodiments, the NADP+ dependent aldehyde dehydrogenase is aldB(NP_418045) from E. coli.

In one embodiment, overexpression of an enzyme to increase intracellularlevels of a coenzyme comprises coupling supplementation of aco-substrate and overexpression of the enzyme. In one embodiment, theoverexpression of an enzyme coupled with supplementation of aco-substrate of that enzyme increase flux through a biochemical pathway.In one embodiment, an NAD+ or NADP+ dependent alcohol dehydrogenase isexpressed with a co-substrate. In certain embodiments, an alcoholdehydrogenase is expressed with an isopropanol co-substrate. In oneembodiment, an NAD+ or NADP+ dependent glucose dehydrogenase isexpressed with a co-substrate. In certain embodiments, a glucosedehydrogenase is expressed with a glucose co-substrate.

In one embodiment, the expression of a transhydrogenase is increased tointerconvert NADH and NADPH. In some embodiments, the transhydrogenaseis a pyridine nucleotide transhydrogenase. In some embodiments, thepyridine nucleotide transhydrogenase is from bacteria. In certainembodiments, the pyridine nucleotide transhydrogenase is pntAB (betasubunit: NP_416119; alpha subunit: NP_416120) from E. coli. In someembodiments, the pyridine nucleotide transhydrogenase is from human. Incertain embodiments, the pyridine nucleotide transhydrogenase is NNT(NP_036475) from Homo sapiens. In certain embodiments, the pyridinenucleotide transhydrogenase is from Solanum tuberosum. In certainembodiments, the pyridine nucleotide transhydrogenase is from Spinaceaoleracea.

In some embodiments, it may be useful to increase the expression ofendogenous or exogenous proteins to induce endoplasmic reticulum (ER)membrane proliferation. In some embodiments, the induction ofendoplasmic reticulum membrane proliferation can improve production offatty alcohols, aldehydes, or acetates. In one embodiment, theexpression of an inactivated HMG-CoA reductase(hydroxymethylglutaryl-CoA reductase) containing one or more ER facingloops is increased. In certain embodiments, the one or more loops isbetween transmembrane domains 6 and 7 of an inactivated HMG-CoAreductase. In some embodiments, the inactivated HMG-CoA reductasecomprises an inactivated protein or chimera which codes for the first500 amino acids or a subsequence of the first 500 amino acids ofYarrowia lipolytica YALI0E04807p. In other embodiments, the inactivatedHMG-CoA reductase comprises an inactivated protein or chimera whichcodes for the first 522 amino acids or a subsequence of the first 522amino acids of HMG1 from Saccharomyces cerevisiae (NP_013636.1). Inother embodiments, the inactivated HMG-CoA reductase comprises aninactivated protein or chimera which codes for the first 522 amino acidsor a subsequence of the first 522 amino acids of HMG2 from Saccharomycescerevisiae (NP_013555.1). In some embodiments, the expression of one ormore regulatory proteins is increased to improve production of fattyalcohols, aldehydes, or acetates. In certain embodiments, the regulatoryprotein comprises HAC1 transcription factor from Saccharomycescerevisiae (NP_116622.1). In certain embodiments, the regulatory proteincomprises HAC1 transcription factor from Yarrowia lipolytica(YALI0B12716p).

Increased synthesis or accumulation can be accomplished by, for example,overexpression of nucleic acids encoding one or more of theabove-described a fatty alcohol pathway enzymes. Overexpression of afatty alcohol pathway enzyme or enzymes can occur, for example, throughincreased expression of an endogenous gene or genes, or through theexpression, or increased expression, of an exogenous gene or genes.Therefore, naturally occurring organisms can be readily modified togenerate non-natural, fatty alcohol producing microorganisms throughoverexpression of one or more nucleic acid molecules encoding a fattyalcohol biosynthetic pathway enzyme. In addition, a non-naturallyoccurring organism can be generated by mutagenesis of an endogenous genethat results in an increase in activity of an enzyme in the fattyalcohol biosynthetic pathways.

Equipped with the present disclosure, the skilled artisan will be ableto readily construct the recombinant microorganisms described herein, asthe recombinant microorganisms of the disclosure can be constructedusing methods well known in the art as exemplified above to exogenouslyexpress at least one nucleic acid encoding a fatty alcohol pathwayenzyme in sufficient amounts to produce a fatty alcohol.

Methods for constructing and testing the expression levels of anon-naturally occurring fatty alcohol-producing host can be performed,for example, by recombinant and detection methods well known in the art.Such methods can be found described in, for example, Sambrook et al.,Molecular Cloning: A Laboratory Manual, Third Ed., Cold Spring HarborLaboratory, New York (2001); Ausubo et al., Current Protocols inMolecular Biology, John Wiley and Sons, Baltimore, Md. (1999).

A variety of mechanisms known in the art can be used to express, oroverexpress, exogenous or endogenous genes. For example, an expressionvector or vectors can be constructed to harbor one or more fatty alcoholbiosynthetic pathway enzyme encoding nucleic acids as exemplified hereinoperably linked to expression control sequences functional in the hostorganism. Expression vectors applicable for use in the microbial hostorganisms of the invention include, for example, plasmids, phagevectors, viral vectors, episomes and artificial chromosomes, includingvectors and selection sequences or markers operable for stableintegration into a host chromosome. Selectable marker genes also can beincluded that, for example, provide resistance to antibiotics or toxins,complement auxotrophic deficiencies, or supply critical nutrients not inthe culture media. Expression control sequences can include constitutiveand inducible promoters, transcription enhancers, transcriptionterminators, and the like which are well known in the art. When two ormore exogenous encoding nucleic acids are to be co-expressed, bothnucleic acids can be inserted, for example, into a single expressionvector or in separate expression vectors. For single vector expression,the encoding nucleic acids can be operationally linked to one commonexpression control sequence or linked to different expression controlsequences, such as one inducible promoter and one constitutive promoter.The transformation of exogenous nucleic acid sequences involved in ametabolic or synthetic pathway can be confirmed using methods well knownin the art.

Expression control sequences are known in the art and include, forexample, promoters, enhancers, polyadenylation signals, transcriptionterminators, internal ribosome entry sites (IRES), and the like, thatprovide for the expression of the polynucleotide sequence in a hostcell. Expression control sequences interact specifically with cellularproteins involved in transcription (Maniatis et al., Science, 236:1237-1245 (1987)). Exemplary expression control sequences are describedin, for example, Goeddel, Gene Expression Technology: Methods inEnzymology, Vol. 185, Academic Press, San Diego, Calif. (1990).

In various embodiments, an expression control sequence may be operablylinked to a polynucleotide sequence. By “operably linked” is meant thata polynucleotide sequence and an expression control sequence(s) areconnected in such a way as to permit gene expression when theappropriate molecules (e.g., transcriptional activator proteins) arebound to the expression control sequence(s). Operably linked promotersare located upstream of the selected polynucleotide sequence in terms ofthe direction of transcription and translation. Operably linkedenhancers can be located upstream, within, or downstream of the selectedpolynucleotide.

In some embodiments, the recombinant microorganism is manipulated todelete, disrupt, mutate, and/or reduce the activity of one or moreendogenous enzymes that catalyzes a reaction in a pathway that competeswith the biosynthesis pathway for the production of a mono- orpoly-unsaturated C₆-C₂₄ fatty alcohol, aldehyde, or acetate.

In some embodiments, the recombinant microorganism is manipulated todelete, disrupt, mutate, and/or reduce the activity of one or moreendogenous enzymes that catalyzes the conversion of a fatty acid into aω-hydroxyfatty acid. In some such embodiments, the enzymes that catalyzethe conversion of a fatty acid into a ω-hydroxyfatty acid are selectedfrom the group consisting of XP_504406, XP_504857, XP_504311, XP_500855,XP_500856, XP_500402, XP_500097, XP_501748, XP_500560, XP_501148,XP_501667, XP_500273, BAA02041, CAA39366, CAA39367, BAA02210, BAA02211,BAA02212, BAA02213, BAA02214, AA073952, AAO73953. AAO73954. AAO073955,AAO73956, AAO73958,AAO73959,AAO73969,AAO73961,AAO73957,XP_002546278,

BAM49649, AAB80867, AAB17462, ADL27534, AAU24352, AAA87602, CAA34612,ABM17701, AAA25760, CAB51047, AAC82967, WP_011027348, or homologsthereof.

In other embodiments, the recombinant microorganism is manipulated todelete, disrupt, mutate, and/or reduce the activity of one or moreendogenous enzymes that catalyzes the conversion of a fatty acyl-CoAinto α,β-enoyl-CoA. In some such embodiments, the enzymes that catalyzethe conversion of a fatty acyl-CoA into α,β-enoyl-CoA are selected fromthe group consisting of CAA04659, CAA04660, CAA04661, CAA04662,CAA04663, CAG79214, AAA34322, AAA34361, AAA34363, CAA29901, BAA04761,AAA34891, AAB08643, CAB15271, BAN55749, CAC44516, ADK16968, AEI37634,WP_000973047, WP_025433422, WP_035184107, WP_026484842, CEL80920,WP_026818657, WP_005293707, WP_005883960, or homologs thereof.

In some embodiments, the recombinant microorganism is manipulated todelete, disrupt, mutate, and/or reduce the activity of one or moreproteins involved in peroxisome biogenesis. In such embodiments, the oneor more proteins involved in peroxisome biogenesis are selected from thegroup consisting of XP_505754, XP_501986, XP_501311, XP_504845,XP_503326, XP_504029, XP_002549868, XP_002547156, XP_002545227,XP_002547350, XP_002546990, EIW11539, EIW08094, EIW11472, EIW09743,EIW0828, or homologs thereof.

In some embodiments, the recombinant microorganism is manipulated todelete, disrupt, mutate, and/or reduce the activity of one or moreendogenous enzymes that catalyzes a reaction in a pathway that competeswith the biosynthesis pathway for one or more unsaturated fatty acyl-CoAintermediates. In one embodiment, the one or more endogenous enzymescomprise one or more diacylglycerol acyltransferases. In the context ofa recombinant yeast microorganism, the recombinant yeast microorganismis engineered to delete, disrupt, mutate, and/or reduce the activity ofone or more diacylglycerol acyltransferases selected from the groupconsisting of YALI0E32769g, YALI0D07986g and CTRG 06209, or homologthereof. In another embodiment, the one or more endogenous enzymescomprise one or more glycerolphospholipid acyltransferases. In thecontext of a recombinant yeast microorganism, the recombinant yeastmicroorganism is engineered to delete, disrupt, mutate, and/or reducethe activity of one or more glycerolphospholipid acyltransferasesselected from the group consisting of YALI0E16797g and CTG_04390, orhomolog thereof. In another embodiment, the one or more endogenousenzymes comprise one or more acyl-CoA/sterol acyltransferases. In thecontext of a recombinant yeast microorganism, the recombinant yeastmicroorganism is engineered to delete, disrupt, mutate, and/or reducethe activity of one or more acyl-CoA/sterol acyltransferases selectedfrom the group consisting of YALI0F06578g, CTRG 01764 and CTRG 01765, orhomolog thereof.

In another embodiment, the recombinant microorganism is manipulated todelete, disrupt, mutate, and/or reduce the activity of one or moreendogenous enzymes that catalyzes a reaction in a pathway that oxidizesfatty aldehyde intermediates. In one embodiment, the one or moreendogenous enzymes comprise one or more fatty aldehyde dehydrogenases.In the context of a recombinant yeast microorganism, the recombinantyeast microorganism is engineered to delete, disrupt, mutate, and/orreduce the activity of one or more fatty aldehyde dehydrogenasesselected from the group consisting of YALI0A17875g, YALI0E15400g,YALI0B01298g, YALI0F23793g, CTRG 05010 and CTRG 04471, or homologthereof.

In another embodiment, the recombinant microorganism is manipulated todelete, disrupt, mutate, and/or reduce the activity of one or moreendogenous enzymes that catalyzes a reaction in a pathway that consumesfatty acetate products. In one embodiment, the one or more endogenousenzymes comprise one or more sterol esterases. In the context of arecombinant yeast microorganism, the recombinant yeast microorganism isengineered to delete, disrupt, mutate, and/or reduce the activity of oneor more sterol esterases selected from the group consisting ofYALI0E32035g, YALI0E00528g, CTRG 01138, CTRG 01683 and CTRG 04630, orhomolog thereof. In another embodiment, the one or more endogenousenzymes comprise one or more triacylglycerol lipases. In the context ofa recombinant yeast microorganism, the recombinant yeast microorganismis engineered to delete, disrupt, mutate, and/or reduce the activity ofone or more triacylglycerol lipases selected from the group consistingof YALI0D17534g, YALI0F10010g, CTRG 00057 and CTRG 06185, or homologthereof. In another embodiment, the one or more endogenous enzymescomprise one or more monoacylglycerol lipases. In the context of arecombinant yeast microorganism, the recombinant yeast microorganism isengineered to delete, disrupt, mutate, and/or reduce the activity of oneor more monoacylglycerol lipases selected from the group consisting ofYALI0C14520g, CTRG 03360 and CTRG 05049, or homolog thereof. In anotherembodiment, the one or more endogenous enzymes comprise one or moreextracellular lipases. In the context of a recombinant yeastmicroorganism, the recombinant yeast microorganism is engineered todelete, disrupt, mutate, and/or reduce the activity of one or moreextracellular lipases selected from the group consisting ofYALI0A20350g, YALI0D19184g, YALI0B09361g, CTRG 05930, CTRG 04188, CTRG02799, CTRG 03052 and CTRG 03885, or homolog thereof.

In another embodiment, the recombinant microorganism is manipulated todelete, disrupt, mutate, and/or reduce the activity of one or moreendogenous reductase or desaturase enzymes that interferes with theunsaturated C₆-C₂₄ fatty alcohol, aldehyde, or acetate, i.e., catalyzesthe conversion of a pathway substrate or product into an unwantedby-product.

Chemical Conversion of Product from Microorganism Synthesis

The present disclosure describes chemical conversions that can be usedto convert a product synthesized by recombinant microorganism into adown-stream product.

In some embodiments, an unsaturated fatty alcohol, aldehyde, acetate, orcarboxylic acid produced by a microorganism can undergo subsequentchemical conversion to produce a pheromone, fragrance, flavor, polymer,or polymer intermediate. Non-limiting examples of chemicaltransformations include esterification, metathesis, and polymerization.

Unsaturated fatty carboxylic acids can be esterified by methods known inthe art. For example, Fischer esterification can be used to covert afatty carboxylic acid to a corresponding fatty ester. See, e.g., Komura,K. et al., Synthesis 2008. 3407-3410.

Elongation of the carbon chain can be performed by known methods tocovert an unsaturated fatty alcohol into an elongated derivativethereof. Olefin metastasis catalysts can be performed to increase thenumber of carbons on the fatty carbon chain and impart Z or Estereochemistry on the corresponding unsaturated product.

In general, any metathesis catalyst stable under the reaction conditionsand nonreactive with functional groups on the fatty substrate (e.g.,alcohol, ester, carboxylic acid, aldehyde, or acetate) can be used withthe present disclosure. Such catalysts are, for example, those describedby Grubbs (Grubbs, R.H., “Synthesis of large and small molecules usingolefin metathesis catalysts.” PMSE Prepr., 2012), herein incorporated byreference in its entirety. Depending on the desired isomer of theolefin, as cis-selective metathesis catalyst may be used, for exampleone of those described by Shahane et al. (Shahane, S., et al.ChemCatChem, 2013. 5(12): p. 3436-3459), herein incorporated byreference in its entirety. Specific catalysts 1-5 exhibitingcis-selectivity are shown below (Scheme 1) and have been describedpreviously (Khan, R. K., et al. J. Am. Chem. Soc., 2013. 135(28): p.10258-61; Hartung, J. et al. J. Am. Chem. Soc., 2013. 135(28): p.10183-5.; Rosebrugh, L. E., et al. J. Am. Chem. Soc., 2013. 135(4): p.1276-9.; Marx, V. M., et al. J. Am. Chem. Soc., 2013. 135(1): p. 94-7.;Herbert, M. B., et al. Angew. Chem. Int. Ed. Engl., 2013. 52(1): p.310-4; Keitz, B. K., et al. J. Am. Chem. Soc., 2012. 134(4): p. 2040-3.;Keitz, B. K., et al. J. Am. Chem. Soc., 2012. 134(1): p. 693-9.; Endo,K. et al. J. Am. Chem. Soc., 2011. 133(22): p. 8525-7).

Additional Z-selective catalysts are described in (Cannon and Grubbs2013; Bronner et al. 2014; Hartung et al. 2014; Pribisko et al. 2014;Quigley and Grubbs 2014) and are herein incorporated by reference intheir entirety. Due to their excellent stability and functional grouptolerance, in some embodiments metathesis catalysts include, but are notlimited to, neutral ruthenium or osmium metal carbene complexes thatpossess metal centers that are formally in the +2 oxidation state, havean electron count of 16, are penta-coordinated, and are of the generalformula LL′AA′M=CRbRc or LL′AA′M=(C=)nCRbRc (Pederson and Grubbs 2002);wherein

-   -   M is ruthenium or osmium;    -   L and L′ are each independently any neutral electron donor        ligand and selected from phosphine, sulfonated phosphine,        phosphite, phosphinite, phosphonite, arsine, stibnite, ether,        amine, amide, imine, sulfoxide, carboxyl, nitrosyl, pyridine,        thioether, or heterocyclic carbenes; and    -   A and A′ are anionic ligands independently selected from        halogen, hydrogen, C₁-C₂₀ alkyl, aryl, C₁-C₂₀ alkoxide,        aryloxide, C₂-C₂₀ alkoxycarbonyl, arylcarboxylate, C₁-C₂₀        carboxylate, arylsulfonyl, C₁-C₂₀ alkylsulfonyl, C₁-C₂₀        alkylsulfinyl; each ligand optionally being substituted with        C₁-C₅ alkyl, halogen, C₁-C₅ alkoxy; or with a phenyl group that        is optionally substituted with halogen, C₁-C₅ alkyl, or C₁-C₅        alkoxy; and A and A′ together may optionally comprise a        bidentate ligand; and    -   R_(b) and R_(c) are independently selected from hydrogen, C₁-C₂₀        alkyl, aryl, C₁-C₂₀ carboxylate, C₁-C₂₀ alkoxy, aryloxy, C₁-C₂₀        alkoxycarbonyl, C₁-C₂₀ alkylthio, C₁-C₂₀ alkylsulfonyl and        C₁-C₂₀ alkylsulfinyl, each of R_(b) and R_(c) optionally        substituted with C₁-C₅ alkyl, halogen, C₁-C₅ alkoxy or with a        phenyl group that is optionally substituted with halogen, C₁-C₅        alkyl, or C₁-C₅ alkoxy.

Other metathesis catalysts such as “well defined catalysts” can also beused. Such catalysts include, but are not limited to, Schrock'smolybdenum metathesis catalyst, 2,6-diisopropylphenylimidoneophylidenemolybdenum (VI) bis(hexafluoro-t-butoxide), described byGrubbs et al. (Tetrahedron 1998, 54: 4413-4450) and Basset's tungstenmetathesis catalyst described by Couturier, J. L. et al. (Angew. Chem.Int. Ed. Engl. 1992, 31: 628).

Catalysts useful in the methods of the disclosure also include thosedescribed by Peryshkov, et al. J. Am. Chem. Soc. 2011, 133: 20754-20757;Wang, et al. Angewandte Chemie, 2013, 52: 1939-1943; Yu, et al. J. Am.Chem. Soc., 2012, 134: 2788-2799; Halford. Chem. Eng. News, 2011, 89(45): 11; Yu, et al. Nature, 2011, 479: 88-93; Lee. Nature, 2011, 471:452-453; Meek, et al. Nature, 2011: 471, 461-466; Flook, et al. J. Am.Chem. Soc. 2011, 133: 1784-1786; Zhao, et al. Org Lett., 2011, 13(4):784-787; Ondi, et al. “High activity, stabilized formulations, efficientsynthesis and industrial use of Mo- and W-based metathesis catalysts”XiMo Technology Updates, 2015:http://www.ximo-inc.com/files/ximo/uploads/download/Summary_3.11.15.pdf;Schrock, et al. Macromolecules, 2010: 43, 7515-7522; Peryshkov, et al.Organometallics 2013: 32, 5256-5259; Gerber, et al. Organometallics2013: 32, 5573-5580; Marinescu, et al. Organometallics 2012: 31,6336-6343; Wang, et al. Angew. Chem. Int. Ed. 2013: 52, 1939-1943; Wang,et al. Chem. Eur. 1 2013: 19, 2726-2740; and Townsend et al. J. Am.Chem. Soc. 2012: 134, 11334-11337.

Catalysts useful in the methods of the disclosure also include thosedescribed in International Pub. No. WO 2014/155185; International Pub.No. WO 2014/172534; U.S. Pat. Appl. Pub. No. 2014/0330018; InternationalPub. No. WO 2015/003815; and International Pub. No. WO 2015/003814.

Catalysts useful in the methods of the disclosure also include thosedescribed in U.S. Pat. Nos. 4,231,947; 4,245,131; 4,427,595; 4,681,956;4,727,215; International Pub. No. WO 1991/009825; U.S. Pat. Nos.5,087,710; 5,142,073; 5,146,033; International Pub. No. WO 1992/019631;U.S. Pat. Nos. 6,121,473; 6,346,652; 8,987,531; U.S. Pat. Appl. Pub. No.2008/0119678; International Pub. No. WO 2008/066754; International Pub.No. WO 2009/094201; U.S. Pat. Appl. Pub. No. 2011/0015430; U.S. Pat.Appl. Pub. No. 2011/0065915; U.S. Pat. Appl. Pub. No. 2011/0077421;International Pub. No. WO 2011/040963; International Pub. No. WO2011/097642; U.S. Pat. Appl. Pub. No. 2011/0237815; U.S. Pat. Appl. Pub.No. 2012/0302710; International Pub. No. WO 2012/167171; U.S. Pat. Appl.Pub. No. 2012/0323000; U.S. Pat. Appl. Pub. No. 2013/0116434;International Pub. No. WO 2013/070725; U.S. Pat. Appl. Pub. No.2013/0274482; U.S. Pat. Appl. Pub. No. 2013/0281706; International Pub.No. WO 2014/139679; International Pub. No. WO 2014/169014; U.S. Pat.Appl. Pub. No. 2014/0330018; and U.S. Pat. Appl. Pub. No. 2014/0378637.

Catalysts useful in the methods of the disclosure also include thosedescribed in International Pub. No. WO 2007/075427; U.S. Pat. Appl. Pub.No. 2007/0282148; International Pub. No. WO 2009/126831; InternationalPub. No. WO 2011/069134; U.S. Pat. Appl. Pub. No. 2012/0123133; U.S.Pat. Appl. Pub. No. 2013/0261312; U.S. Pat. Appl. Pub. No. 2013/0296511;International Pub. No. WO 2014/134333; and U.S. Pat. Appl. Pub. No.2015/0018557.

Catalysts useful in the methods of the disclosure also include those setforth in the following table:

Structure Name

dichloro[1,3-bis(2,6-isopropylphenyl)-2-imidazolidinylidene](benzylidene)(tricyclohex- ylphosphine)ruthenium(II)

dichloro[1,3-bis(2,6-isopropylphenyl)-2- imidazolidinylidene](2-isopropoxyphenylmethylene)ruthenium(II)

dichloro[1,3-Bis(2-methylphenyl)-2-imidazolidinylidene](benzylidene)(tricyclohex- ylphosphine)ruthenium(II)

dichloro[1,3-bis(2-methylphenyl)-2- imidazolidinylidene](2-isopropoxyphenylmethylene)ruthenium(II)

dichloro[1,3-bis(2,4,6-trimethylphenyl)-2-imidazolidinylidene](benzylidene)bis(3- bromopyridine)ruthenium(II)

dichloro[1,3-bis(2,4,6-trimethylphenyl)-2-imidazolidinylidene](3-methyl-2- butenylidene) (tricyclohexylphosphine)ruthenium(II)

dichloro[1,3-bis(2,4,6-trimethylphenyl)-2-imidazolidinylidene][3-(2-pyridinyl) propylidene]ruthenium(II)

dichloro[1,3-bis(2,4,6-trimethylphenyl)-2-imidazolidinylidene][(tricyclohexylphos-phoranyl)methylidene]ruthenium(II) tetrafluoroborate

dichloro(3-methyl-2-butenylidene)bis(tricyclohexylphosphine)ruthenium(II)

dichloro(3-methyl-2-butenylidene)bis(tricyclopentylphosphine)ruthenium(II)

dichloro(tricyclohexylphosphine)[(tricyclohex-ylphosphoranyl)methylidene]ruthenium(II) tetrafluoroborate

bis(tricyclohexylphosphine) benzylidine ruthenium(IV) dichloride

[1,3-bis-(2,4,6-trimethylphenyl)-2-imidazolidinylidene]dichloro(phenylmethylene)(tricyclohexylphosphine)ruthenium

(1,3-bis-(2,4,6-trimethylphenyl)-2- imidazolidinylidene)dichloro(o-isopropoxyphenylmethylene)ruthenium

dichloro(o- isopropoxyphenylmethylene)(tricyclohexyl-phosphine)ruthenium(II)

[2-(1-methylethoxy-O)phenylmethyl-C](nitrato-O,O′){rel-(2R,5R,7R)-adamantane-2,1-diyl[3-(2,4,6-trimethylphenyl)-1-imidazolidinyl-2-ylidene]}ruthenium

Catalysts useful in the methods of the disclosure also include thosedescribed in U.S. Pat. Appl. Pub. No. 2008/0009598; U.S. Pat. Appl. Pub.No. 2008/0207911; U.S. Pat. Appl. Pub. No. 2008/0275247; U.S. Pat. Appl.Pub. No. 2011/0040099; U.S. Pat. Appl. Pub. No. 2011/0282068; and U.S.Pat. Appl. Pub. No. 2015/0038723.

Catalysts useful in the methods of the disclosure include thosedescribed in International Pub. No. WO 2007/140954; U.S. Pat. Appl. Pub.No. 2008/0221345; International Pub. No. WO 2010/037550; U.S. Pat. Appl.Pub. No. 2010/0087644; U.S. Pat. Appl. Pub. No. 2010/0113795; U.S. Pat.Appl. Pub. No. 2010/0174068; International Pub. No. WO 2011/091980;International Pub. No. WO 2012/168183; U.S. Pat. Appl. Pub. No.2013/0079515; U.S. Pat. Appl. Pub. No. 2013/0144060; U.S. Pat. Appl.Pub. No. 2013/0211096; International Pub. No. WO 2013/135776;International Pub. No. WO 2014/001291; International Pub. No. WO2014/067767; U.S. Pat. Appl. Pub. No. 2014/0171607; and U.S. Pat. Appl.Pub. No. 2015/0045558.

The catalyst is typically provided in the reaction mixture in asub-stoichiometric amount (e.g., catalytic amount). In certainembodiments, that amount is in the range of about 0.001 to about 50 mol% with respect to the limiting reagent of the chemical reaction,depending upon which reagent is in stoichiometric excess. In someembodiments, the catalyst is present in less than or equal to about 40mol % relative to the limiting reagent. In some embodiments, thecatalyst is present in less than or equal to about 30 mol % relative tothe limiting reagent. In some embodiments, the catalyst is present inless than about 20 mol %, less than about 10 mol %, less than about 5mol %, less than about 2.5 mol %, less than about 1 mol %, less thanabout 0.5 mol %, less than about 0.1 mol %, less than about 0.015 mol %,less than about 0.01 mol %, less than about 0.0015 mol %, or less,relative to the limiting reagent. In some embodiments, the catalyst ispresent in the range of about 2.5 mol % to about 5 mol %, relative tothe limiting reagent. In some embodiments, the reaction mixture containsabout 0.5 mol % catalyst. In the case where the molecular formula of thecatalyst complex includes more than one metal, the amount of thecatalyst complex used in the reaction may be adjusted accordingly.

In some cases, the methods described herein can be performed in theabsence of solvent (e.g., neat). In some cases, the methods can includethe use of one or more solvents. Examples of solvents that may besuitable for use in the disclosure include, but are not limited to,benzene, p-cresol, toluene, xylene, diethyl ether, glycol, diethylether, petroleum ether, hexane, cyclohexane, pentane, methylenechloride, chloroform, carbon tetrachloride, dioxane, tetrahydrofuran(THF), dimethyl sulfoxide, dimethylformamide, hexamethyl-phosphorictriamide, ethyl acetate, pyridine, triethylamine, picoline, and thelike, as well as mixtures thereof. In some embodiments, the solvent isselected from benzene, toluene, pentane, methylene chloride, and THF. Incertain embodiments, the solvent is benzene.

In some embodiments, the method is performed under reduced pressure.This may be advantageous in cases where a volatile byproduct, such asethylene, may be produced during the course of the metathesis reaction.For example, removal of the ethylene byproduct from the reaction vesselmay advantageously shift the equilibrium of the metathesis reactiontowards formation of the desired product. In some embodiments, themethod is performed at a pressure of about less than 760 torr. In someembodiments, the method is performed at a pressure of about less than700 torr. In some embodiments, the method is performed at a pressure ofabout less than 650 torr. In some embodiments, the method is performedat a pressure of about less than 600 torr. In some embodiments, themethod is performed at a pressure of about less than 550 torr. In someembodiments, the method is performed at a pressure of about less than500 torr. In some embodiments, the method is performed at a pressure ofabout less than 450 torr. In some embodiments, the method is performedat a pressure of about less than 400 torr. In some embodiments, themethod is performed at a pressure of about less than 350 torr. In someembodiments, the method is performed at a pressure of about less than300 torr. In some embodiments, the method is performed at a pressure ofabout less than 250 torr. In some embodiments, the method is performedat a pressure of about less than 200 torr. In some embodiments, themethod is performed at a pressure of about less than 150 torr. In someembodiments, the method is performed at a pressure of about less than100 torr. In some embodiments, the method is performed at a pressure ofabout less than 90 torr. In some embodiments, the method is performed ata pressure of about less than 80 torr. In some embodiments, the methodis performed at a pressure of about less than 70 torr. In someembodiments, the method is performed at a pressure of about less than 60torr. In some embodiments, the method is performed at a pressure ofabout less than 50 torr. In some embodiments, the method is performed ata pressure of about less than 40 torr. In some embodiments, the methodis performed at a pressure of about less than 30 torr. In someembodiments, the method is performed at a pressure of about less than 20torr. In some embodiments, the method is performed at a pressure ofabout 20 torr.

In some embodiments, the method is performed at a pressure of about 19torr. In some embodiments, the method is performed at a pressure ofabout 18 torr. In some embodiments, the method is performed at apressure of about 17 torr. In some embodiments, the method is performedat a pressure of about 16 torr. In some embodiments, the method isperformed at a pressure of about 15 torr. In some embodiments, themethod is performed at a pressure of about 14 torr. In some embodiments,the method is performed at a pressure of about 13 torr. In someembodiments, the method is performed at a pressure of about 12 torr. Insome embodiments, the method is performed at a pressure of about 11torr. In some embodiments, the method is performed at a pressure ofabout 10 torr. In some embodiments, the method is performed at apressure of about 10 torr. In some embodiments, the method is performedat a pressure of about 9 torr. In some embodiments, the method isperformed at a pressure of about 8 torr. In some embodiments, the methodis performed at a pressure of about 7 torr. In some embodiments, themethod is performed at a pressure of about 6 torr. In some embodiments,the method is performed at a pressure of about 5 torr. In someembodiments, the method is performed at a pressure of about 4 torr. Insome embodiments, the method is performed at a pressure of about 3 torr.In some embodiments, the method is performed at a pressure of about 2torr. In some embodiments, the method is performed at a pressure ofabout 1 torr. In some embodiments, the method is performed at a pressureof less than about 1 torr.

In some embodiments, the two metathesis reactants are present inequimolar amounts. In some embodiments, the two metathesis reactants arenot present in equimolar amounts. In certain embodiments, the tworeactants are present in a molar ratio of about 20:1, 19:1, 18:1, 17:1,16:1, 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1,3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12,1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, or 1:20. In certainembodiments, the two reactants are present in a molar ratio of about10:1. In certain embodiments, the two reactants are present in a molarratio of about 7:1. In certain embodiments, the two reactants arepresent in a molar ratio of about 5:1. In certain embodiments, the tworeactants are present in a molar ratio of about 2:1. In certainembodiments, the two reactants are present in a molar ratio of about1:10. In certain embodiments, the two reactants are present in a molarratio of about 1:7. In certain embodiments, the two reactants arepresent in a molar ratio of about 1:5. In certain embodiments, the tworeactants are present in a molar ratio of about 1:2.

In general, the reactions with many of the metathesis catalystsdisclosed herein provide yields better than 15%, better than 50%, betterthan 75%, or better than 90%. In addition, the reactants and productsare chosen to provide at least a 5° C. difference, a greater than 20° C.difference, or a greater than 40° C. difference in boiling points.Additionally, the use of metathesis catalysts allows for much fasterproduct formation than byproduct, it is desirable to run these reactionsas quickly as practical. In particular, the reactions are performed inless than about 24 hours, less than 12 hours, less than 8 hours, or lessthan 4 hours.

One of skill in the art will appreciate that the time, temperature andsolvent can depend on each other, and that changing one can requirechanging the others to prepare the pyrethroid products and intermediatesin the methods of the disclosure. The metathesis steps can proceed at avariety of temperatures and times. In general, reactions in the methodsof the disclosure are conducted using reaction times of several minutesto several days. For example, reaction times of from about 12 hours toabout 7 days can be used. In some embodiments, reaction times of 1-5days can be used. In some embodiments, reaction times of from about 10minutes to about 10 hours can be used. In general, reactions in themethods of the disclosure are conducted at a temperature of from about0° C. to about 200° C. For example, reactions can be conducted at15-100° C. In some embodiments, reaction can be conducted at 20-80° C.In some embodiments, reactions can be conducted at 100-150° C.

Unsaturated fatty esters can be reduced using a suitable reducing agentwhich selectively reduces the ester to the corresponding aldehyde oralcohol but does not reduce the double bond. An unsaturated fatty estercan be reduced to the corresponding unsaturated fatty aldehyde usingdi-isobutyl aluminum halide (DIBAL) or Vitride®. The unsaturated fattyaldehyde can be reduced to the corresponding fatty alcohol with, e.g.,DIBAL or Vitride®. In some embodiments, the unsaturated fatty ester canbe reduced to the corresponding fatty alcohol using A1H3 or9-Borabicyclo(3.3.1)nonane (9-BBN). (See Galatis, P. Encyclopedia ofReagents for Organic Synthesis. 2001. New York: John Wiley & Sons; andCarey & Sunderburg. Organic Chemistry, Part B: Reactions and Synthesis,5^(th) edition. 2007. New York. Springer Sciences.)

Pheromone Compositions and Uses Thereof

As described above, products made via the methods described herein arepheromones. Pheromones prepared according to the methods of theinvention can be formulated for use as insect control compositions. Thepheromone compositions can include a carrier, and/or be contained in adispenser. The carrier can be, but is not limited to, an inert liquid orsolid.

Examples of solid carriers include but are not limited to fillers suchas kaolin, bentonite, dolomite, calcium carbonate, talc, powderedmagnesia, Fuller's earth, wax, gypsum, diatomaceous earth, rubber,plastic, China clay, mineral earths such as silicas, silica gels,silicates, attaclay, limestone, chalk, loess, clay, dolomite, calciumsulfate, magnesium sulfate, magnesium oxide, ground synthetic materials,fertilizers such as ammonium sulfate, ammonium phosphate, ammoniumnitrate, thiourea and urea, products of vegetable origin such as cerealmeals, tree bark meal, wood meal and nutshell meal, cellulose powders,attapulgites, montmorillonites, mica, vermiculites, synthetic silicasand synthetic calcium silicates, or compositions of these.

Examples of liquid carriers include, but are not limited to, water;alcohols, such as ethanol, butanol or glycol, as well as their ethers oresters, such as methylglycol acetate; ketones, such as acetone,cyclohexanone, methylethyl ketone, methylisobutylketone, or isophorone;alkanes such as hexane, pentane, or heptanes; aromatic hydrocarbons,such as xylenes or alkyl naphthalenes; mineral or vegetable oils;aliphatic chlorinated hydrocarbons, such as trichloroethane or methylenechloride; aromatic chlorinated hydrocarbons, such as chlorobenzenes;water-soluble or strongly polar solvents such as dimethylformamide,dimethyl sulfoxide, or N-methylpyrrolidone; liquefied gases; waxes, suchas beeswax, lanolin, shellac wax, carnauba wax, fruit wax (such asbayberry or sugar cane wax) candelilla wax, other waxes such asmicrocrystalline, ozocerite, ceresin,or montan; salts such asmonoethanolamine salt, sodium sulfate, potassium sulfate, sodiumchloride, potassium chloride, sodium acetate, ammonium hydrogen sulfate,ammonium chloride, ammonium acetate, ammonium formate, ammonium oxalate,ammonium carbonate, ammonium hydrogen carbonate, ammonium thiosulfate,ammonium hydrogen diphosphate, ammonium dihydrogen monophosphate,ammonium sodium hydrogen phosphate, ammonium thiocyanate, ammoniumsulfamate or ammonium carbamateand mixtures thereof. Baits or feedingstimulants can also be added to the carrier.

Synergist

In some embodiments, the pheromone composition is combined with anactive chemical agent such that a synergistic effect results. Thesynergistic effect obtained by the taught methods can be quantifiedaccording to Colby's formula (i.e. (E)=X+Y−(X*Y/100). See Colby, R. S.,“Calculating Synergistic and Antagonistic Responses of HerbicideCombinations”, 1967 Weeds, vol. 15, pp. 20-22, incorporated herein byreference in its entirety. Thus, by “synergistic” is intended acomponent which, by virtue of its presence, increases the desired effectby more than an additive amount. The pheromone compositions andadjuvants of the present methods can synergistically increase theeffectiveness of agricultural active compounds and also agriculturalauxiliary compounds.

Thus, in some embodiments, a pheromone composition can be formulatedwith a synergist. The term, “synergist,” as used herein, refers to asubstance that can be used with a pheromone for reducing the amount ofthe pheromone dose or enhancing the effectiveness of the pheromone forattracting at least one species of insect. The synergist may or may notbe an independent attractant of an insect in the absence of a pheromone.

In some embodiments, the synergist is a volatile phytochemical thatattracts at least one species of Lepidoptera. The term, “phytochemical,”as used herein, means a compound occurring naturally in a plant species.In a particular embodiment, the synergist is selected from the groupcomprising β-caryophyllene, iso-caryophyllene, α-humulene, inalool,Z3-hexenol/yl acetate, β-farnesene, benzaldehyde, phenylacetaldehyde,and combinations thereof.

The pheromone composition can contain the pheromone and the synergist ina mixed or otherwise combined form, or it may contain the pheromone andthe synergist independently in a non-mixed form.

Insecticide

The pheromone composition can include one or more insecticides. In oneembodiment, the insecticides are chemical insecticides known to oneskilled in the art. Examples of the chemical insecticides include one ormore of pyrethoroid or organophosphorus insecticides, including but arenot limited to, cyfluthrin, permethrin, cypermethrin, bifinthrin,fenvalerate, flucythrinate, azinphosmethyl, methyl parathion,buprofezin, pyriproxyfen, flonicamid, acetamiprid, dinotefuran,clothianidin, acephate, malathion, quinolphos, chloropyriphos,profenophos, bendiocarb, bifenthrin, chlorpyrifos, cyfluthrin, diazinon,pyrethrum, fenpropathrin, kinoprene, insecticidal soap or oil,neonicotinoids, diamides, avermectin and derivatives, spinosad andderivatives, azadirachtin, pyridalyl, and mixtures thereof.

In another embodiment, the insecticides are one or more biologicalinsecticides known to one skilled in the art. Examples of the biologicalinsecticides include, but are not limited to, azadirachtin (neem oil),toxins from natural pyrethrins, Bacillus thuringiencis and Beauveriabassiana, viruses (e.g., CYD-X™, CYD-X HP™, Germstar™, Madex HP™ andSpod-X™), peptides (Spear-T™, Spear-P™, and Spear-C™)

In another embodiment, the insecticides are insecticides that target thenerve and muscle. Examples include acetylcholinesterase (AChE)inhibitors, such as carbamates (e.g., methomyl and thiodicarb) andorganophosphates (e.g., chlorpyrifos) GABA-gated chloride channelantagonists, such as cyclodiene organochlorines (e.g., endosulfan) andphenylpyrazoles (e.g., fipronil), sodium channel modulators, such aspyrethrins and pyrethroids (e.g., cypermethrin and λ-cyhalothrin),nicotinic acetylcholine receptor (nAChR) agonists, such asneonicotinoids (e.g., acetamiprid, tiacloprid, thiamethoxam), nicotinicacetylcholine receptor (nAChR) allosteric modulators, such as spinosyns(e.g., spinose and spinetoram), chloride channel activators, such asavermectins and milbemycins (e.g., abamectin, emamectin benzoate),Nicotinic acetylcholine receptor (nAChR) blockers, such as bensultap andcartap, voltage dependent sodium channel blockers, such as indoxacarband metaflumizone, ryanodine receptor modulator, such as diamides (e.g.dhlorantraniliprole and flubendiamide). In another embodiment, theinsecticides are insecticides that target respiration. Examples includechemicals that uncouple oxidative phosphorylation via disruption of theproton gradient, such as chlorfenapyr, and mitochondrial complex Ielectron transport inhibitors.

In another embodiment, the insecticides are insecticides that targetmidgut. Examples include microbial disruptors of insect midgutmembranes, such as Bacillus thuringiensis and Bacillus sphaericus.

In another embodiment, the insecticides are insecticides that targetgrowth and development. Examples include juvenile hormone mimics, suchas juvenile hormone analogues (e.g. fenoxycarb), inhibitors of chitinbiosynthesis, Type 0, such as benzoylureas (e.g., flufenoxuron,lufenuron, and novaluron), and ecdysone receptor agonists, such asdiacylhydrazines (e.g., methoxyfenozide and tebufenozide)

Stabilizer

According to another embodiment of the disclosure, the pheromonecomposition may include one or more additives that enhance the stabilityof the composition. Examples of additives include, but are not limitedto, fatty acids and vegetable oils, such as for example olive oil,soybean oil, corn oil, safflower oil, canola oil, and combinationsthereof.

Filler

According to another embodiment of the disclosure, the pheromonecomposition may include one or more fillers. Examples of fillersinclude, but are not limited to, one or more mineral clays (e.g.,attapulgite). In some embodiments, the attractant-composition mayinclude one or more organic thickeners. Examples of such thickenersinclude, but are not limited to, methyl cellulose, ethyl cellulose, andany combinations thereof.

Solvent

According to another embodiment, the pheromone compositions of thepresent disclosure can include one or more solvents. Compositionscontaining solvents are desirable when a user is to employ liquidcompositions which may be applied by brushing, dipping, rolling,spraying, or otherwise applying the liquid compositions to substrates onwhich the user wishes to provide a pheromone coating (e.g., a lure). Insome embodiments, the solvent(s) to be used is/are selected so as tosolubilize, or substantially solubilize, the one or more ingredients ofthe pheromone composition. Examples of solvents include, but are notlimited to, water, aqueous solvent (e.g., mixture of water and ethanol),ethanol, methanol, chlorinated hydrocarbons, petroleum solvents,turpentine, xylene, and any combinations thereof.

In some embodiments, the pheromone compositions of the presentdisclosure comprise organic solvents. Organic solvents are used mainlyin the formulation of emulsifiable concentrates, ULV formulations, andto a lesser extent granular formulations. Sometimes mixtures of solventsare used. In some embodiments, the present disclosure teaches the use ofsolvents including aliphatic paraffinic oils such as kerosene or refinedparaffins. In other embodiments, the present disclosure teaches the useof aromatic solvents such as xylene and higher molecular weightfractions of C9 and C10 aromatic solvents. In some embodiments,chlorinated hydrocarbons are useful as co-solvents to preventcrystallization when the formulation is emulsified into water. Alcoholsare sometimes used as co-solvents to increase solvent power.

Solubilizing Agent

In some embodiments, the pheromone compositions of the presentdisclosure comprise solubilizing agents. A solubilizing agent is asurfactant, which will form micelles in water at concentrations abovethe critical micelle concentration. The micelles are then able todissolve or solubilize water-insoluble materials inside the hydrophobicpart of the micelle. The types of surfactants usually used forsolubilization are non-ionics: sorbitan monooleates; sorbitan monooleateethoxylates; and methyl oleate esters.

Binder

According to another embodiment of the disclosure, the pheromonecomposition may include one or more binders. Binders can be used topromote association of the pheromone composition with the surface of thematerial on which said composition is coated. In some embodiments, thebinder can be used to promote association of another additive (e.g.,insecticide, insect growth regulators, and the like) to the pheromonecomposition and/or the surface of a material. For example, a binder caninclude a synthetic or natural resin typically used in paints andcoatings. These may be modified to cause the coated surface to befriable enough to allow insects to bite off and ingest the components ofthe composition (e.g., insecticide, insect growth regulators, and thelike), while still maintaining the structural integrity of the coating.

Non-limiting examples of binders include polyvinylpyrrolidone, polyvinylalcohol, partially hydrolyzed polyvinyl acetate, carboxymethylcellulose,starch, vinylpyrrolidone/vinyl acetate copolymers and polyvinyl acetate,or compositions of these; lubricants such as magnesium stearate, sodiumstearate, talc or polyethylene glycol, or compositions of these;antifoams such as silicone emulsions, long-chain alcohols, phosphoricesters, acetylene diols, fatty acids or organofluorine compounds, andcomplexing agents such as: salts of ethylenediaminetetraacetic acid(EDTA), salts of trinitrilotriacetic acid or salts of polyphosphoricacids, or compositions of these.

In some embodiments, the binder also acts a filler and/or a thickener.Examples of such binders include, but are not limited to, one or more ofshellac, acrylics, epoxies, alkyds, polyurethanes, linseed oil, tungoil, and any combinations thereof.

Surface-Active Agents

In some embodiments, the pheromone compositions comprise surface-activeagents. In some embodiments, the surface-active agents are added toliquid agricultural compositions. In other embodiments, thesurface-active agents are added to solid formulations, especially thosedesigned to be diluted with a carrier before application. Thus, in someembodiments, the pheromone compositions comprise surfactants.Surfactants are sometimes used, either alone or with other additives,such as mineral or vegetable oils as adjuvants to spray-tank mixes toimprove the biological performance of the pheromone on the target. Thesurface-active agents can be anionic, cationic, or nonionic incharacter, and can be employed as emulsifying agents, wetting agents,suspending agents, or for other purposes. In some embodiments, thesurfactants are non-ionics such as: alky ethoxylates, linear aliphaticalcohol ethoxylates, and aliphatic amine ethoxylates. Surfactantsconventionally used in the art of formulation and which may also be usedin the present formulations are described, in McCutcheon's Detergentsand Emulsifiers Annual, MC Publishing Corp., Ridgewood, N. J., 1998, andin Encyclopedia of Surfactants, Vol. I-III, Chemical Publishing Co., NewYork, 1980-81. In some embodiments, the present disclosure teaches theuse of surfactants including alkali metal, alkaline earth metal orammonium salts of aromatic sulfonic acids, for example, ligno-, phenol-,naphthalene- and dibutylnaphthalenesulfonic acid, and of fatty acids ofarylsulfonates, of alkyl ethers, of lauryl ethers, of fatty alcoholsulfates and of fatty alcohol glycol ether sulfates, condensates ofsulfonated naphthalene and its derivatives with formaldehyde,condensates of naphthalene or of the naphthalenesulfonic acids withphenol and formaldehyde, condensates of phenol or phenolsulfonic acidwith formaldehyde, condensates of phenol with formaldehyde and sodiumsulfite, polyoxyethylene octylphenyl ether, ethoxylated isooctyl-,octyl- or nonylphenol, tributylphenyl polyglycol ether, alkylarylpolyether alcohols, isotridecyl alcohol, ethoxylated castor oil,ethoxylated triarylphenols, salts of phosphatedtriarylphenolethoxylates, lauryl alcohol polyglycol ether acetate,sorbitol esters, lignin-sulfite waste liquors or methylcellulose, orcompositions of these.

In some embodiments, the present disclosure teaches other suitablesurface-active agents, including salts of alkyl sulfates, such asdiethanolammonium lauryl sulfate; alkylarylsulfonate salts, such ascalcium dodecylbenzenesulfonate; alkylphenol-alkylene oxide additionproducts, such as nonylphenol-C18 ethoxylate; alcohol-alkylene oxideaddition products, such as tridecyl alcohol-C16 ethoxylate; soaps, suchas sodium stearate; alkylnaphthalene-sulfonate salts, such as sodiumdibutyl-naphthalenesulfonate; dialkyl esters of sulfosuccinate salts,such as sodium di(2-ethylhexyl)sulfosuccinate; sorbitol esters, such assorbitol oleate; quaternary amines, such as lauryl trimethylammoniumchloride; polyethylene glycol esters of fatty acids, such aspolyethylene glycol stearate; block copolymers of ethylene oxide andpropylene oxide; salts of mono and dialkyl phosphate esters; vegetableoils such as soybean oil, rapeseed/canola oil, olive oil, castor oil,sunflower seed oil, coconut oil, corn oil, cottonseed oil, linseed oil,palm oil, peanut oil, safflower oil, sesame oil, tung oil and the like;and esters of the above vegetable oils, particularly methyl esters.

Wetting Agents

In some embodiments, the pheromone compositions comprise wetting agents.A wetting agent is a substance that when added to a liquid increases thespreading or penetration power of the liquid by reducing the interfacialtension between the liquid and the surface on which it is spreading.Wetting agents are used for two main functions in agrochemicalformulations: during processing and manufacture to increase the rate ofwetting of powders in water to make concentrates for soluble liquids orsuspension concentrates; and during mixing of a product with water in aspray tank or other vessel to reduce the wetting time of wettablepowders and to improve the penetration of water into water-dispersiblegranules. In some embodiments, examples of wetting agents used in thepheromone compositions of the present disclosure, including wettablepowders, suspension concentrates, and water-dispersible granuleformulations are: sodium lauryl sulphate; sodium dioctylsulphosuccinate; alkyl phenol ethoxylates; and aliphatic alcoholethoxylates.

Dispersing Agent

In some embodiments, the pheromone compositions of the presentdisclosure comprise dispersing agents. A dispersing agent is a substancewhich adsorbs onto the surface of particles and helps to preserve thestate of dispersion of the particles and prevents them fromreaggregating. In some embodiments, dispersing agents are added topheromone compositions of the present disclosure to facilitatedispersion and suspension during manufacture, and to ensure theparticles redisperse into water in a spray tank. In some embodiments,dispersing agents are used in wettable powders, suspension concentrates,and water-dispersible granules. Surfactants that are used as dispersingagents have the ability to adsorb strongly onto a particle surface andprovide a charged or steric barrier to re-aggregation of particles. Insome embodiments, the most commonly used surfactants are anionic,non-ionic, or mixtures of the two types.

In some embodiments, for wettable powder formulations, the most commondispersing agents are sodium lignosulphonates. In some embodiments,suspension concentrates provide very good adsorption and stabilizationusing polyelectrolytes, such as sodium naphthalene sulphonateformaldehyde condensates. In some embodiments, tristyrylphenolethoxylated phosphate esters are also used. In some embodiments, such asalkylarylethylene oxide condensates and EO-PO block copolymers aresometimes combined with anionics as dispersing agents for suspensionconcentrates.

Polymeric Surfactant

In some embodiments, the pheromone compositions of the presentdisclosure comprise polymeric surfactants. In some embodiments, thepolymeric surfactants have very long hydrophobic ‘backbones’ and a largenumber of ethylene oxide chains forming the ‘teeth’ of a ‘comb’surfactant. In some embodiments, these high molecular weight polymerscan give very good long-term stability to suspension concentrates,because the hydrophobic backbones have many anchoring points onto theparticle surfaces. In some embodiments, examples of dispersing agentsused in pheromone compositions of the present disclosure are: sodiumlignosulphonates; sodium naphthalene sulphonate formaldehydecondensates; tristyrylphenol ethoxylate phosphate esters; aliphaticalcohol ethoxylates; alky ethoxylates; EO-PO block copolymers; and graftcopolymers.

Emulsifying Agent

In some embodiments, the pheromone compositions of the presentdisclosure comprise emulsifying agents. An emulsifying agent is asubstance, which stabilizes a suspension of droplets of one liquid phasein another liquid phase. Without the emulsifying agent the two liquidswould separate into two immiscible liquid phases. In some embodiments,the most commonly used emulsifier blends include alkylphenol oraliphatic alcohol with 12 or more ethylene oxide units and theoil-soluble calcium salt of dodecylbenzene sulphonic acid. A range ofhydrophile-lipophile balance (“HLB”) values from 8 to 18 will normallyprovide good stable emulsions. In some embodiments, emulsion stabilitycan sometimes be improved by the addition of a small amount of an EO-POblock copolymer surfactant.

Gelling Agent

In some embodiments, the pheromone compositions comprise gelling agents.Thickeners or gelling agents are used mainly in the formulation ofsuspension concentrates, emulsions, and suspoemulsions to modify therheology or flow properties of the liquid and to prevent separation andsettling of the dispersed particles or droplets. Thickening, gelling,and anti-settling agents generally fall into two categories, namelywater-insoluble particulates and water-soluble polymers. It is possibleto produce suspension concentrate formulations using clays and silicas.In some embodiments, the pheromone compositions comprise one or morethickeners including, but not limited to: montmorillonite, e.g.bentonite; magnesium aluminum silicate; and attapulgite. In someembodiments, the present disclosure teaches the use of polysaccharidesas thickening agents. The types of polysaccharides most commonly usedare natural extracts of seeds and seaweeds or synthetic derivatives ofcellulose. Some embodiments utilize xanthan and some embodiments utilizecellulose. In some embodiments, the present disclosure teaches the useof thickening agents including, but are not limited to: guar gum; locustbean gum; carrageenam; alginates; methyl cellulose; sodium carboxymethylcellulose (SCMC); hydroxyethyl cellulose (HEC). In some embodiments, thepresent disclosure teaches the use of other types of anti-settlingagents such as modified starches, polyacrylates, polyvinyl alcohol, andpolyethylene oxide. Another good anti-settling agent is xanthan gum.

Anti-Foam Agent

In some embodiments, the presence of surfactants, which lowerinterfacial tension, can cause water-based formulations to foam duringmixing operations in production and in application through a spray tank.Thus, in some embodiments, in order to reduce the tendency to foam,anti-foam agents are often added either during the production stage orbefore filling into bottles/spray tanks. Generally, there are two typesof anti-foam agents, namely silicones and nonsilicones. Silicones areusually aqueous emulsions of dimethyl polysiloxane, while thenonsilicone anti-foam agents are water-insoluble oils, such as octanoland nonanol, or silica. In both cases, the function of the anti-foamagent is to displace the surfactant from the air-water interface.

Preservative

In some embodiments, the pheromone compositions comprise a preservative.

Additional Active Agent

According to another embodiment of the disclosure, the pheromonecomposition may include one or more insect feeding stimulants. Examplesof insect feeding stimulants include, but are not limited to, crudecottonseed oil, fatty acid esters of phytol, fatty acid esters ofgeranyl geraniol, fatty acid esters of other plant alcohols, plantextracts, and combinations thereof.

According to another embodiment of the disclosure, the pheromonecomposition may include one or more insect growth regulators (“IGRs”).IGRs may be used to alter the growth of the insect and produce deformedinsects. Examples of insect growth regulators include, for example,dimilin.

According to another embodiment of the disclosure, theattractant-composition may include one or more insect sterilants thatsterilize the trapped insects or otherwise block their reproductivecapacity, thereby reducing the population in the following generation.In some situations allowing the sterilized insects to survive andcompete with non-trapped insects for mates is more effective thankilling them outright.

Sprayable Compositions

In some embodiments, the pheromone compositions disclosed herein can beformulated as a sprayable composition (i.e., a sprayable pheromonecomposition). An aqueous solvent can be used in the sprayablecomposition, e.g., water or a mixture of water and an alcohol, glycol,ketone, or other water-miscible solvent. In some embodiments, the watercontent of such mixture is at least about 10%, at least about 20%, atleast about 30%, at least about 40%, 50%, at least about 60%, at leastabout 70%, at least about 80%, or at least about 90%. In someembodiments, the sprayable composition is concentrate, i.e. aconcentrated suspension of the pheromone, and other additives (e.g., awaxy substance, a stabilizer, and the like) in the aqueous solvent, andcan be diluted to the final use concentration by addition of solvent(e.g., water).

In some embodiments, the a waxy substance can be used as a carrier forthe pheromone and its positional isomer in the sprayable composition.The waxy substance can be, e.g., a biodegradable wax, such as bees wax,carnauba wax and the like, candelilla wax (hydrocarbon wax), montan wax,shellac and similar waxes, saturated or unsaturated fatty acids, such aslauric, palmitic, oleic or stearic acid, fatty acid amides and esters,hydroxylic fatty acid esters, such as hydroxyethyl or hydroxypropylfatty acid esters, fatty alcohols, and low molecular weight polyesterssuch as polyalkylene succinates.

In some embodiments, a stabilizer can be used with the sprayablepheromone compositions. The stabilizer can be used to regulate theparticle size of concentrate and/or to allow the preparation of a stablesuspension of the pheromone composition. In some embodiments, thestabilizer is selected from hydroxylic and/or ethoxylated polymers.Examples include ethylene oxide and propylene oxide copolymer,polyalcohols, including starch, maltodextrin and other solublecarbohydrates or their ethers or esters, cellulose ethers, gelatin,polyacrylic acid and salts and partial esters thereof and the like. Inother embodiments, the stabilizer can include polyvinyl alcohols andcopolymers thereof, such as partly hydrolyzed polyvinyl acetate. Thestabilizer may be used at a level sufficient to regulate particle sizeand/or to prepare a stable suspension, e.g., between 0.1% and 15% of theaqueous solution.

In some embodiments, a binder can be used with the sprayable pheromonecompositions. In some embodiments, the binder can act to furtherstabilize the dispersion and/or improve the adhesion of the sprayeddispersion to the target locus (e.g., trap, lure, plant, and the like).The binder can be polysaccharide, such as an alginate, cellulosederivative (acetate, alkyl, carboxymethyl, hydroxyalkyl), starch orstarch derivative, dextrin, gum (arabic, guar, locust bean, tragacanth,carrageenan, and the like), sucrose, and the like. The binder can alsobe a non-carbohydrate, water-soluble polymer such as polyvinylpyrrolidone, or an acidic polymer such as polyacrylic acid orpolymethacrylic acid, in acid and/or salt form, or mixtures of suchpolymers.

Microencapsulated Pheromones

In some embodiments, the pheromone compositions disclosed herein can beformulated as a microencapsulated pheromone, such as disclosed inIll'lchev, A L et al., J. Econ. Entomol. 2006; 99(6):2048-54; andStelinki, L L et al., J. Econ. Entomol. 2007; 100(4):1360-9.Microencapsulated pheromones (MECs) are small droplets of pheromoneenclosed within polymer capsules. The capsules control the release rateof the pheromone into the surrounding environment, and are small enoughto be applied in the same method as used to spray insecticides. Theeffective field longevity of the microencapsulated pheromoneformulations can range from a few days to slightly more than a week,depending on inter alia climatic conditions, capsule size and chemicalproperties.

Slow-Release Formulation

Pheromone compositions can be formulated so as to provide slow releaseinto the atmosphere, and/or so as to be protected from degradationfollowing release. For example, the pheromone compositions can beincluded in carriers such as microcapsules, biodegradable flakes andparaffin wax-based matrices. Alternatively, the pheromone compositioncan be formulated as a slow release sprayable.

In certain embodiments, the pheromone composition may include one ormore polymeric agents known to one skilled in the art. The polymericagents may control the rate of release of the composition to theenvironment. In some embodiments, the polymeric attractant-compositionis impervious to environmental conditions. The polymeric agent may alsobe a sustained-release agent that enables the composition to be releasedto the environment in a sustained manner.

Examples of polymeric agents include, but are not limited to,celluloses, proteins such as casein, fluorocarbon-based polymers,hydrogenated rosins, lignins, melamine, polyurethanes, vinyl polymerssuch as polyvinyl acetate (PVAC), polycarbonates, polyvinylidenedinitrile, polyamides, polyvinyl alcohol (PVA), polyamide-aldehyde,polyvinyl aldehyde, polyesters, polyvinyl chloride (PVC), polyethylenes,polystyrenes, polyvinylidene, silicones, and combinations thereof.Examples of celluloses include, but are not limited to, methylcellulose,ethyl cellulose, cellulose acetate, cellulose acetate-butyrate,cellulose acetate-propionate, cellulose propionate, and combinationsthereof.

Other agents which can be used in slow-release or sustained-releaseformulations include fatty acid esters (such as a sebacate, laurate,palmitate, stearate or arachidate ester) or a fatty alcohols (such asundecanol, dodecanol, tridecanol, tridecenol, tetradecanol,tetradecenol, tetradecadienol, pentadecanol, pentadecenol, hexadecanol,hexadecenol, hexadecadienol, octadecenol and octadecadienol).

Pheromones prepared according to the methods of the invention, as wellas compositions containing the pheromones, can be used to control thebehavior and/or growth of insects in various environments. Thepheromones can be used, for example, to attract or repel male or femaleinsects to or from a particular target area. The pheromones can be usedto attract insects away from vulnerable crop areas. The pheromones canalso be used example to attract insects as part of a strategy for insectmonitoring, mass trapping, lure/attract-and-kill or mating disruption.

Lures

The pheromone compositions of the present disclosure may be coated on orsprayed on a lure, or the lure may be otherwise impregnated with apheromone composition.

Traps

The pheromone compositions of the disclosure may be used in traps, suchas those commonly used to attract any insect species, e.g., insects ofthe order Lepidoptera. Such traps are well known to one skilled in theart, and are commonly used in many states and countries in insecteradication programs. In one embodiment, the trap includes one or moresepta, containers, or storage receptacles for holding the pheromonecomposition. Thus, in some embodiments, the present disclosure providesa trap loaded with at least one pheromone composition. Thus, thepheromone compositions of the present disclosure can be used in trapsfor example to attract insects as part of a strategy for insectmonitoring, mass trapping, mating disruption, or lure/attract and killfor example by incorporating a toxic substance into the trap to killinsects caught.

Mass trapping involves placing a high density of traps in a crop to beprotected so that a high proportion of the insects are removed beforethe crop is damaged. Lure/attract-and-kill techniques are similar exceptonce the insect is attracted to a lure, it is subjected to a killingagent. Where the killing agent is an insecticide, a dispenser can alsocontain a bait or feeding stimulant that will entice the insects toingest an effective amount of an insecticide. The insecticide may be aninsecticide known to one skilled in the art. The insecticide may bemixed with the attractant-composition or may be separately present in atrap. Mixtures may perform the dual function of attracting and killingthe insect.

Such traps may take any suitable form, and killing traps need notnecessarily incorporate toxic substances, the insects being optionallykilled by other means, such as drowning or electrocution. Alternatively,the traps can contaminate the insect with a fungus or virus that killsthe insect later. Even where the insects are not killed, the trap canserve to remove the male insects from the locale of the female insects,to prevent breeding.

It will be appreciated by a person skilled in the art that a variety ofdifferent traps are possible. Suitable examples of such traps includewater traps, sticky traps, and one-way traps. Sticky traps come in manyvarieties. One example of a sticky trap is of cardboard construction,triangular or wedge-shaped in cross-section, where the interior surfacesare coated with a non-drying sticky substance. The insects contact thesticky surface and are caught. Water traps include pans of water anddetergent that are used to trap insects. The detergent destroys thesurface tension of the water, causing insects that are attracted to thepan, to drown in the water. One-way traps allow an insect to enter thetrap but prevent it from exiting. The traps of the disclosure can becolored brightly, to provide additional attraction for the insects.

In some embodiments, the pheromone traps containing the composition maybe combined with other kinds of trapping mechanisms. For example, inaddition to the pheromone composition, the trap may include one or moreflorescent lights, one or more sticky substrates and/or one or morecolored surfaces for attracting moths. In other embodiments, thepheromone trap containing the composition may not have other kinds oftrapping mechanisms.

The trap may be set at any time of the year in a field. Those of skillin the art can readily determine an appropriate amount of thecompositions to use in a particular trap, and can also determine anappropriate density of traps/acre of crop field to be protected.

The trap can be positioned in an area infested (or potentially infested)with insects. Generally, the trap is placed on or close to a tree orplant. The aroma of the pheromone attracts the insects to the trap. Theinsects can then be caught, immobilized and/or killed within the trap,for example, by the killing agent present in the trap.

Traps may also be placed within an orchard to overwhelm the pheromonesemitted by the females, so that the males simply cannot locate thefemales. In this respect, a trap need be nothing more than a simpleapparatus, for example, a protected wickable to dispense pheromone.

The traps of the present disclosure may be provided in made-up form,where the compound of the disclosure has already been applied. In suchan instance, depending on the half-life of the compound, the compoundmay be exposed, or may be sealed in conventional manner, such as isstandard with other aromatic dispensers, the seal only being removedonce the trap is in place.

Alternatively, the traps may be sold separately, and the compound of thedisclosure provided in dispensable format so that an amount may beapplied to trap, once the trap is in place. Thus, the present disclosuremay provide the compound in a sachet or other dispenser.

Dispenser

Pheromone compositions can be used in conjunction with a dispenser forrelease of the composition in a particular environment. Any suitabledispenser known in the art can be used. Examples of such dispensersinclude but are not limited to, aerosol emitters, hand-applieddispensers, bubble caps comprising a reservoir with a permeable barrierthrough which pheromones are slowly released, pads, beads, tubes rods,spirals or balls composed of rubber, plastic, leather, cotton, cottonwool, wood or wood products that are impregnated with the pheromonecomposition. For example, polyvinyl chloride laminates, pellets,granules, ropes or spirals from which the pheromone compositionevaporates, or rubber septa. One of skill in the art will be able toselect suitable carriers and/or dispensers for the desired mode ofapplication, storage, transport or handling.

In another embodiment, a device may be used that contaminates the maleinsects with a powder containing the pheromone substance itself. Thecontaminated males then fly off and provide a source of matingdisruption by permeating the atmosphere with the pheromone substance, orby attracting other males to the contaminated males, rather than to realfemales.

Behavior Modification

Pheromone compositions prepared according to the methods disclosedherein can be used to control or modulate the behavior of insects. Insome embodiments, the behavior of the target insect can be modulated ina tunable manner inter alia by varying the ratio of the pheromone to thepositional isomer in the composition such that the insect is attractedto a particular locus but does not contact said locus or such the insectin fact contacts said locus. Thus, in some embodiments, the pheromonescan be used to attract insects away from vulnerable crop areas.Accordingly, the disclosure also provides a method for attractinginsects to a locus. The method includes administering to a locus aneffective amount of the pheromone composition.

The method of mating disruption may include periodically monitoring thetotal number or quantity of the trapped insects. The monitoring may beperformed by counting the number of insects trapped for a predeterminedperiod of time such as, for example, daily, Weekly, bi-Weekly, monthly,once-in-three months, or any other time periods selected by the monitor.Such monitoring of the trapped insects may help estimate the populationof insects for that particular period, and thereby help determine aparticular type and/or dosage of pest control in an integrated pestmanagement system. For example, a discovery of a high insect populationcan necessitate the use of methods for removal of the insect. Earlywarning of an infestation in a new habitat can allow action to be takenbefore the population becomes unmanageable. Conversely, a discovery of alow insect population can lead to a decision that it is sufficient tocontinue monitoring the population. Insect populations can be monitoredregularly so that the insects are only controlled when they reach acertain threshold. This provides cost-effective control of the insectsand reduces the environmental impact of the use of insecticides.

Mating Disruption

Pheromones prepared according to the methods of the disclosure can alsobe used to disrupt mating. Mating disruption is a pest managementtechnique designed to control insect pests by introducing artificialstimuli (e.g., a pheromone composition as disclosed herein) thatconfuses the insects and disrupts mating localization and/or courtship,thereby preventing mating and blocking the reproductive cycle.

In many insect species of interest to agriculture, such as those in theorder Lepidoptera, females emit an airborne trail of a specific chemicalblend constituting that species' sex pheromone. This aerial trail isreferred to as a pheromone plume. Males of that species use theinformation contained in the pheromone plume to locate the emittingfemale (known as a “calling” female). Mating disruption exploits themale insects' natural response to follow the plume by introducing asynthetic pheromone into the insects' habitat, which is designed tomimic the sex pheromone produced by the female insect. Thus, in someembodiments, the synthetic pheromone utilized in mating disruption is asynthetically derived pheromone composition comprising a pheromonehaving a chemical structure of a sex pheromone and a positional isomerthereof which is not produced by the target insect.

The general effect of mating disruption is to confuse the male insectsby masking the natural pheromone plumes, causing the males to follow“false pheromone trails” at the expense of finding mates, and affectingthe males' ability to respond to “calling” females. Consequently, themale population experiences a reduced probability of successfullylocating and mating with females, which leads to the eventual cessationof breeding and collapse of the insect infestation

Strategies of mating disruption include confusion, trail-masking andfalse-trail following. Constant exposure of insects to a highconcentration of a pheromone can prevent male insects from responding tonormal levels of the pheromone released by female insects. Trail-maskinguses a pheromone to destroy the trail of pheromones released by females.False-trail following is carried out by laying numerous spots of apheromone in high concentration to present the male with many falsetrails to follow. When released in sufficiently high quantities, themale insects are unable to find the natural source of the sex pheromones(the female insects) so that mating cannot occur.

In some embodiments, a wick or trap may be adapted to emit a pheromonefor a period at least equivalent to the breeding season(s) of the midge,thus causing mating disruption. If the midge has an extended breedingseason, or repeated breeding season, the present disclosure provides awick or trap capable of emitting pheromone for a period of time,especially about two weeks, and generally between about 1 and 4 weeksand up to 6 weeks, which may be rotated or replaced by subsequentsimilar traps. A plurality of traps containing the pheromone compositionmay be placed in a locus, e.g., adjacent to a crop field. The locationsof the traps, and the height of the traps from ground may be selected inaccordance with methods known to one skilled in the art.

Alternatively, the pheromone composition may be dispensed fromformulations such as microcapsules or twist-ties, such as are commonlyused for disruption of the mating of insect pests.

Attract and Kill

The attract and kill method utilizes an attractant, such as a sexpheromone, to lure insects of the target species to an insecticidalchemical, surface, device, etc., for mass-killing and ultimatepopulation suppression, and can have the same effect as mass-trapping.For instance, when a synthetic female sex pheromone is used to lure malepests, e.g., moths, in an attract-and-kill strategy, a large number ofmale moths must be killed over extended periods of time to reducematings and reproduction, and ultimately suppress the pest population.The attract-and-kill approach may be a favorable alternative tomass-trapping because no trap-servicing or other frequent maintenance isrequired. In various embodiments described herein, a recombinantmicroorganism can co-express (i) a pathway for production of an insectpheromone and (ii) a protein, peptide, oligonucleotide, or smallmolecule which is toxic to the insect. In this way, the recombinantmicroorganism can co-produce substances suitable for use in anattract-and-kill approach.

As will be apparent to one of skill in the art, the amount of apheromone or pheromone composition used for a particular application canvary depending on several factors such as the type and level ofinfestation; the type of composition used; the concentration of theactive components; how the composition is provided, for example, thetype of dispenser used; the type of location to be treated; the lengthof time the method is to be used for; and environmental factors such astemperature, wind speed and direction, rainfall and humidity. Those ofskill in the art will be able to determine an effective amount of apheromone or pheromone composition for use in a given application.

As used herein, an “effective amount” means that amount of the disclosedpheromone composition that is sufficient to affect desired results. Aneffective amount can be administered in one or more administrations. Forexample, an effective amount of the composition may refer to an amountof the pheromone composition that is sufficient to attract a giveninsect to a given locus. Further, an effective amount of the compositionmay refer to an amount of the pheromone composition that is sufficientto disrupt mating of a particular insect population of interest in agiven locality.

EXAMPLES Prophetic Example 1. Production of Pheromones Products fromEnzymatically-Derived Gondoic Acid Through Metathesis and ChemicalConversion

This prophetic example illustrates that different fatty acids can beused as a starting material for the biosynthetic production of apheromone or pheromone precursor. The product obtained from thebiosynthetic process disclosed herein can be subject to further chemicalconversions to generate different products.

Enzymatic two carbon elongation of oleic acid yields gondoic acid. Afteresterification, gondoic fatty acid methyl ester (FAME) can thenconverted via Z-selective olefin metathesis into C16 and C18 FAMEproducts containing a C11 unsaturation. Upon reduction of the ester,aldehyde and fatty alcohol pheromone materials can be produced.Acetylation of the fatty alcohol product can generate the correspondingfatty acetate pheromones. Additionally, gondoic acid can be directlyconverted into C20 fatty aldehyde, alcohol and acetate pheromonesthrough application of the same chemical transformation of enzymaticallymodified oleic acid.

Prophetic Example 2: Tailored Synthetic Blends

This prophetic example illustrates that the recombinant microorganismsdisclosed herein can be used to create synthetic blends of insectpheromones.

As shown in the scheme in FIG. 40, using tetradecyl-ACP (14:ACP), ablend of E- and Z-tetradecenyl acetate (E11-14:OAc and Z11-14:OAC)pheromones can be produced with the recombinant microorganism. Thisblend is produced by a variety of insects, e.g., Choristoneura roseceana(a moth of the Tortricidae family).

Similarly, using hexadecyl-ACP (16:ACP), a blend of Z- and E hexadecenylacetate pheromones (E11-16:OAc and Z11-16:OAc) can be produced with therecombinant microorganism.

The microorganism can be engineered with different desaturases, or otherenzymes such as reductases, etc. to produce the desired blend ofpheromones. One blend of particular relevance capable of being producedusing the recombinant microorganisms and methods of the instantinvention is a 97:3 ratio of (Z)-11-hexadecenal (Z11-16:Ald) and(Z)-9-hexadecenal (Z9-16:Ald).

Example 3: Expression of Transmembrane Alcohol-Forming Reductases in S.cerevisiae Background and Rationale

Engineering microbial production of insect fatty alcohols from fattyacids entails the functional expression of a synthetic pathway. One suchpathway comprises a transmembrane desaturase, and an alcohol-formingreductase to mediate the conversion of fatty acyl-CoA into regio- andstereospecific unsaturated fatty acyl-CoA, and subsequently into fattyalcohols. A number of genes encoding these enzymes are found in someinsects (as well as some microalgae in the case of fatty alcoholreductase) and can be used to construct the synthetic pathway in yeasts,which are preferred production hosts. A number of transmembranedesaturases and alcohol-forming reductase variants will be screened toidentify ensembles which allow high level synthesis of a single insectfatty alcohol or a blend of fatty alcohols. Additionally, these enzymeswill be screened across multiple hosts (Saccharomyces cerevisiae,Candida tropicalis, and Yarrowia lipolytica) to optimize the searchtoward finding a suitable host for optimum expression of thesetransmembrane proteins.

Summary of Approach

Three alcohol-forming reductases of insect origin were selected.

Nucleic acids encoding the reductases were synthesized (synthons) withcodon optimization for expression in S. cerevisiae.

Each nucleic acid encoding a given reductase was subcloned into anepisomal expression cassette under the Gall promoter.

S. cerevisiae wild-type and beta-oxidation deletion mutant weretransformed with expression constructs.

Heterologous protein was induced by galactose, and functional expressionof the reductases was assessed in vivo via bioconversion ofZ11-hexadecenoic acid into Z11-hexedecenol.

GC-MS analysis was used to identify and quantify metabolites.

Results

Alcohol-forming reductase variants were screened for activity in S.cerevisiae W303 (wild type) and BY4742 ΔPOX1 (beta-oxidation deletionmutant). Z11-hexadecenoic acid was chosen as a substrate in assessingenzyme activity. The in vivo bioconversion assay showed that theexpression of enzyme variants derived from Spodoptera littoralis,Helicoverpa armigera, and Agrotis segetum (Ding, B-J., Lofstedt, C.Analysis of the Agrotis segetum pheromone gland transcriptome in thelight of sex pheromone biosynthesis. BMC Genomics 16:711 (2015)) inW303A conferred Z11-hexadecenol production, and reached up-to ˜37 μM (8mg/L), ˜70 μM (˜16 mg/L), and 11 μM (˜3 mg/L), respectively, within 48 hof protein induction (FIG. 5 and FIG. 6). Biologically-producedZ11-hexadecenol matched authentic Z11-hexadecenol standard (Bedoukian)as determined via GC-MS (FIG. 7). BY4742 ΔPOX1 was also explored as anexpression host since deletion in the key beta-oxidation pathway enzymecould limit the degradation of Z11-hexadecenoic acid. Expressing thereductase variants in the beta-oxidation deletion mutant, however,reduced the product titer when compared to expression in the wild-typehost (FIG. 5). One contributing factor of titer reduction when usingBY4742 ΔPOX1 as a host was the reduction of biomass when compared toW303 (FIG. 8).

Therefore, functional expression of at least two alcohol-formingreductases in S. cerevisiae conferred bioconversion of Z11-hexadecenoicacid into Z11-hexedecenol.

Conclusions

Functional expression of insect transmembrane alcohol-forming reductasein S. cerevisiae was demonstrated. Among the reductases tested, thevariant derived from Helicoverpa armigera is most active towardZ11-hexadecenoic acid.

The bioconversion of other fatty acid substrates can be explored toassess enzyme plasticity.

Materials & Methods Strain Construction and Functional Expression Assay

S. cerevisiae W303 (MATA ura3-1 trp1-1 leu2-3_112 his3-11_15 ade2-1can1-100) and BY4742 (MATa PDX1::kanMX his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0) wereused as expression hosts. DNA sequences which encode fatty alcoholreductase variants were redesigned to optimize expression in S.cerevisiae (SEQ ID NOs: 1-3). Generated synthons (Genscript) were clonedinto pESC-URA vector using BamHI-XhoI sites to facilitate proteinexpression utilizing the Gall promoter. The resulting plasmid constructswere used to transform W303, and positive transformants were selected onCM agar medium (with 2% glucose, and lacking uracil) (Teknova). Toassess functional expression, two positive transformation clones thathave been patched on CM agar medium (with 2% glucose, and lackinguracil) were used to seed CM liquid medium using a 24 deep-well plateformat. To induce protein expression, the overnight cultures that hadbeen grown at 28° C. were then supplemented with galactose, raffinose,and YNB to a final concentration of 2%, 1%, and 6.7 g/L, respectively.Post 24 h of protein induction, the bioconversion substrateZ11-hexadecenoic acid (in ethanol) or heptadecanoic acid (in ethanol)was added to a final concentration of 300 mg/L. Bioconversion assayproceeded for 48 h at 28° C. prior to GC-MS analysis.

Metabolite Extraction and GC-MS Detection

The lipids were extracted according to a modified procedure of Hagströmet al. (2012) (Hagström, A. K., Liénard, M. A., Groot, A. T.,Hedenstrom, E. & Lofstedt, C. Semi-Selective Fatty Acyl Reductases fromFour Heliothine Moths Influence the Specific Pheromone Composition. PLoSOne 7: e37230 (2012)). 1.5 mL-cell culture was transferred to a 15 mLfalcon tube. The cell suspension was acidified with 1 mL 5 N HCl. 5 μLtetradecanedioic acid (10 mM in ethanol) was added as internal standard.The mixture was extracted by adding 1.5 mL hexane, then shaken for 1 hat 37° C., 250 rpm. To facilitate phase separation, the sample wascentrifuged for 10 min at 2000 g. 1 mL of the organic hexane phase wasthen transferred to a 1.5 mL plastic tube. The solvent was removed byheating the sample 30 min at 90° C. After the sample was evaporated todryness, 50 μL of BSTFA (N,O-bis(trimethylsilyl) trifluoroacetamidecontaining 1% of trimethylchlorosilane) was added. The 1.5 mL plastictubes were shaken vigorously two times for 10 s. Prior to the transferinto a screw cap GC glass vial containing a glass insert, the sample wascentrifuged for 1 min (13000 rpm). The vials were capped and heated for30 min at 90° C. The trimethylsilyl-esters, which were generated by thismethod were subsequently analyzed by GC-MS analysis. GC-MS parametersare specified in Table 6. The use of SIM mode (characteristic productand IS ions) increases detection sensitivity by reducing backgroundnoise, allowing detection of the product as low as 2.4 μM (0.6 mg/L). Afurther reduction in the split ratio offers the possibility to furtherincrease the sensitivity for future applications. A Z11-hexadecenolcalibration curve shown in FIG. 9 was used to quantify theZ11-hexadecenol produced from yeasts. The bioconversion of heptadecanoicacid was also tested since the easily distinguished heptadecanol productcould be used to benchmark successful GC-MS runs. However, none of thereductase tested showed any activity toward heptadecanoic acid.

TABLE 6 GC-MS parameters System Agent 6890 N GC, ChemStation G1701EAE.02.01.1177 Column Rtx-5 30 m × 320 μm × 25 μm Pressure = 11.74 psi;Flow = 7.1 mL/min Inlet Heater = 250° C.; Pressure = 11.74 psi; TotalFlow {He} = 19.5 mL/min Carrier He @ 147 cm/sec, 11.74 psi Signal Datarate = 2 Hz/0.1 min Oven 150° C. for 1 min Ramp 12° C./min to 220° C.,hold 3 min Ramp 35° C./min to 300° C., hold 4 min Injection Split, 250°C. Split ratio - 20:1 Detector HP 5973 MSD in SIM mode (m/z: 297.3 and387.3), 100 msec Dwell, EMV mode: Gain factor 1, 3 min solvent delay,8.33 cycles/sec| Sample Injection volume = 1 uL

Example 4: Expression of Transmembrane Desaturases in S. cerevisiaeBackground and Rationale

Engineering microbial production of insect fatty alcohols from fattyacids requires the functional expression of a synthetic pathway. Onesuch pathway comprises a transmembrane desaturase, and analcohol-forming reductase to mediate the conversion of fatty acyl-CoAinto regio- and stereospecific unsaturated fatty acyl-CoA, andsubsequently into fatty alcohols. A number of genes encoding theseenzymes are found in some insects as well as some microalgae. A numberof transmembrane desaturases and alcohol-forming reductase variants willbe screened to identify ensembles which allow high level synthesis of asingle insect fatty alcohol or a blend of fatty alcohols. Additionally,these enzymes will be screened across multiple hosts (Saccharomycescerevisiae, Candida tropicalis, and Yarrowia lipolytica) to optimize thesearch toward finding a suitable host for optimum expression of thesetransmembrane proteins.

Summary of Approach

A small set of desaturases (insect origin: Agrotis segetum, Trichoplusiani, Amyelois transitella, Helicoverpa zea, and marine diatom:Thalassiosira pseudonana) were selected as a test case to explore andestablish functional expression assays, metabolite extraction methods,and analytical chemistry.

A synthetic cassette for expression of the desaturases in S. cerevisiaewas constructed. The cassette consists of the OLE1 promoter region, OLE1N-terminal leader sequence, and VSP13 terminator.

The expression cassette was tested for functionality via expression of aGFP variant. Validation of the cassette allowed its utilization forexploring expression of insect desaturase.

S. cerevisiae ΔOLE1 was transformed with expression constructscontaining heterologous desaturases. Functionality of the desaturaseswas assessed via the ability to rescue growth of ΔOLE1 without exogenoussupplementation of unsaturated fatty acid (UFA). S. cerevisiaedesaturase (OLE1) was used as a positive control of successfulcomplementation.

Functionality of the desaturase was validated via an in vivobioconversion of hexadecanoic acid (palmitic acid) into(Z)-11-hexadecenoic acid (palmitvaccenic acid).

GC-MS analysis was used to identify and quantify metabolites.

Results

Transmembrane desaturase variants were screened in S. cerevisiae. Threevariants were initially tested to explore and establish functionalexpression assays, metabolite extraction methods, and analyticalchemistry. To allow functional expression of these desaturases in S.cerevisiae, an episomal synthetic expression cassette termed pOLE1cassette (FIG. 10) was constructed, which consisted of an OLE1 promoterregion, an N-terminal leader sequence encoding for the first 27 aminoacids of S. cerevisiae OLE1, and a terminator region of VPS13 (a proteininvolved in the protospore membrane formation, the terminator of whichhas been previously characterized to increase heterologous proteinexpression potentially by extending mRNA half-life). The functionalityof the pOLE1 cassette was validated via its ability to express a GFP(FIG. 11A-FIG. 11E). Subsequently, insect desaturase synthons, and yeastOLE1 synthon were cloned into the pOLE1 cassette, and expressed in S.cerevisiae ΔOLE1 strain. This strain was chosen since deletion of theOLE1 allele (which encodes for palmitoyl:CoA/stearoyl:CoA(z)-9-desaturase) allows its utilization as a tool to screen forfunctional insect desaturase. Specifically, an active desaturase wouldallow complementation of growth without requiring exogenoussupplementation of UFAs. Expression of OLE1 using pOLE1 cassettecomplemented growth of ΔOLE1 growth without UFA (FIG. 11A-FIG. 11E);therefore, it serves as a positive control in the complementationassays. When insect desaturases were expressed, we observed that theyrescued ΔOLE1 growth without UFA at varying degree. On rich medium (YPD)agar plate, expression of S. cerevisiae OLE1 conferred the highest levelof growth, followed by T. ni desaturase (FIG. 12A). The latter indicatedthat production of unsaturated fatty acyl:CoA by T. ni desaturase couldact as a surrogate to the missing (Z)-9-hexadecenoyl:CoA biosynthesis inΔOLE1. Expression of T. pesudonana and A. segetum desaturases did notappear to rescue growth on YPD very well (FIG. 12A). When patched onminimal medium (CM-Ura glucose) agar plate, only expression of S.cerevisiae OLE1 and T. ni desaturase rescued ΔOLE1 growth withoutexogenous UFA (FIG. 12B). Expression of T. pseudonana and A. segetumdesaturases did not confer growth of ΔOLE1 on minimal medium agar,suggesting their limited activity in producing UFA (results not shown).Screening a desaturase library in Candida tropicalis identifiedfunctional expression of A. transitella and H. zea desaturases. Whenthese desaturases were expressed in ΔOLE1, they conferred growth withoutUFA on both YPD and CM-Ura glucose media similar to expression of T. nidesaturase (FIG. 12B).

Functional expression of the heterologous desaturases was furthercharacterized via in vivo bioconversion of palmitic acid intoinsect-specific UFA. Post ˜96 h-cultivation in minimal medium containingpalmitic acid, total fatty acid analysis of S. cerevisiae ΔOLE1expressing T. ni desaturase revealed production of a new fatty acidspecies (Z)-11-hexadecenoic acid that is not present in the controlstrain which expresses native yeast OLE1 desaturase (FIG. 13A-FIG. 13B).(Z)-11-hexadecenoic acid is not detected in strains expressing A.segetum, or T. pseudonana desaturase (results not shown). In addition to(Z)-11-hexadecenoic acid, (Z)-9-hexadecenoic acid was also detected inΔOLE1 strain expressing T. ni desaturase (FIG. 13A-FIG. 13B). Under thecultivation condition, C16-fatty acid in the ΔOLE1 expressing T. nidesaturase is composed of approximately 84.7% hexadecanoic acid, 5.6%(Z)-9-hexadecenoic acid and 9.8% (Z)-11-hexadeceneoic acid. Incomparison, the C16 fatty acid fraction of ΔOLE1 expressing OLE1desaturase is composed of approximately 68.6% hexadecanoic acid and31.4% (Z)-9-hexadecenoic acid. (Z)-11-hexadecenoic acid biosynthesis inΔOLE1 expressing T. ni desaturase account for ˜1.5 mg/L. The amount oftotal fatty acids and each fatty acid within this mixture can bequantified. The biologically produced (Z)-11-hexadecenoic acid alsomatch the retention time and fragmentation pattern of authentic standard(Z)-11-hexadecenoic acid (Larodan) as determined by GC-MS (FIG. 14A-FIG.14B). Therefore, the regio- and stereoisomer of the biologicallyproduced (Z)-11-hexadecenoic acid was confirmed. In vivocharacterization of A. transitella and H. zea desaturase can also bedone.

In summary, at least three insect desaturases capable of rescuing growthof S. cerevisiae ΔOLE1 without exogenous supplementation of UFA, i.e.(Z)-9-hexadecenoic acid (palmitoleic acid), were identified.

The extent of growth on rich medium (YPD) of S. cerevisiae ΔOLE1 bearingthe expression construct was in the following order of desaturasecontent: OLE1, T. ni, T pseudonana, and A. segetum.

The extent of growth on minimal medium (CM Glucose w/out uracil) of S.cerevisiae ΔOLE1 bearing the expression construct was in the followingorder of desaturase content: OLE1, T. ni.

Complementation assays using A. transitella and H. zea desaturases werealso done, demonstrating functional expression in Candida tropicalisshown via in vivo bioconversion assay. These desaturases alsocomplemented S. cerevisiae ΔOLE1 growth on rich and minimal media atleast as well as T. ni desaturase.

Expression of T. pseudonana and A. segetum desaturases did not confergrowth of S. cerevisiae ΔOLE1 on minimal medium without UFAs even afteran extended incubation period up to 14 days. No (Z)-11-hexadecenoic acidwas observed in strains harboring T. pseudonana or A. segetumdesaturase.

Conclusions

Functional expression of transmembrane desaturases of insect origin inS. cerevisiae has been achieved.

The activity of a given heterologous desaturase can be assessed from itsability to complement growth of S. cerevisiae ΔOLE1 without exogenouspalmitoleic supplementation, and its ability to convert palmitic acidinto insect pheromone precursors (Z)-11-hexadecenoic acid.

Functional expression and/or activity of insect desaturase in S.cerevisiae varies widely depending on sequence origin. Variants derivedfrom T. ni exhibited the best activity compared to A. segetum and T.pseudonana, as measured by the above criteria.

Desaturases derived from A. transitella and H. zea complemented ΔOLE1 aswell as T. ni desaturase. Bioconversion assays using these desaturasescan be done.

The bioconversion of other fatty acid substrates can be explored toassess enzyme plasticity.

Materials & Methods Strain Construction and Functional Expression Assay

S. cerevisiae ΔOLE1 (MATA OLE1::LEU2 ura3-52 his4) was used as anexpression host. A synthetic expression cassette termed pOLE1 (FIG. 10,SEQ ID NO: 4) which comprises the OLE1 promoter region (SEQ ID NOs: 5and 6), nucleotides encoding for 27 N-terminal amino acids of the OLE1leader sequence (SEQ ID NO: 7), and a VPS13 terminator sequence (SEQ IDNO: 8) was created, and cloned into pESC-URA vector in between SacI andEcoRI sites. To test the functionality of the pOLE1 cassette, Dasher GFPsynthon was inserted in between SpeI and NotI sites to create pOLE1-GFPplasmid. Competent ΔOLE1 was transformed with pOLE1-GFP, and plated onCM-Ura glucose agar plate (Teknova) containing UFA (20 mm CM-URA glucoseagar plate was coated with 100 μL CM-Ura glucose medium containing 1%tergitol, and 3 μL palmitoleic acid). After incubation at 30° C. for 5days, Dasher GFP expression was apparent as displayed by greencoloration of ΔOLE1 transformants. This result showed that the pOLE1cassette was capable of driving heterologous protein expression.Validation of ΔOLE1 complementation was performed by restoring OLE1activity. Specifically, native S. cerevisiae OLE1 synthon was insertedinto pOLE1 cassette devoid of the leader sequence to create pOLE1-OLE1plasmid. After transformation of ΔOLE1, and selection on CM-Ura glucoseagar containing UFA, single colonies were patched onto YPD and CM-Uraglucose without UFA. After incubation at 30° C. for 5 days, growth wasobserved (FIG. 11A-FIG. 11E). As expected, Dasher GFP expression couldnot complement ΔOLE1 growth without UFA (FIG. 11A-FIG. 11E). DNAsequences which encode for desaturase variants were synthesized (toinclude nucleotide changes which remove restriction sites used forcloning purposes), and cloned into pOLE1 using SpeI-NotI sites(Genscript, SEQ ID NOs: 9-13). Complementation assay of ΔOLE1 withinsect desaturases were performed in the same way as with OLE1desaturase.

To assess functional expression, two positive transformation clones thathad been patched on CM-Ura glucose agar medium containing UFA wereinoculated in 1.5 mL CM-Ura glucose liquid medium containing palmiticacid (in ethanol) at a final concentration of 300 mg/L, and with 6.7 g/Lof YNB. For (z)-11-hexadecenoic isomer confirmation, a 20 mL culture wasgenerated. Bioconversion assay proceeded for 96 h at 28° C. prior toGC-MS analysis.

Metabolite Extraction and GC-MS Detection

Total lipid composition as well as the (Z)-11-hexadecenoic acidquantification was based on modified procedures by Moss et al. (1982)(Moss, C. W., Shinoda, T. & Samuels, J. W. Determination of cellularfatty acid compositions of various yeasts by gas-liquid chromatography.J. Clin. Microbiol. 16: 1073-1079 (1982)) and Yousuf et al (2010)(Yousuf, A., Sannino, F., Addorisio, V. & Pirozzi, D. MicrobialConversion of Olive Oil Mill Wastewaters into Lipids Suitable forBiodiesel Production. J. Agric. Food Chem. 58: 8630-8635 (2010)). Thepelleted cells (in 1.5 mL plastic tubes), usually about 10 mg to 80 mg,were resuspended in methanol containing 5% (w/w) of sodium hydroxide.The alkaline cell suspension was transferred into a 1.8 mL screw-capGC-vial. The mixture was heated for 1 h in the heat block at 90° C.Prior to acidification with 400 2.5 N HCl the vial was allowed to coolto room temperature. 500 μL chloroform containing 1 mM heptadecanoicwere added and the mixture was shaken vigorously, then both aqueous andorganic phase were transferred into a 1.5 mL plastic tube. The mixturewas centrifuged at 13,000 rpm, afterwards 450 μL of the organic phasewere transferred into a new 1.5 mL plastic tube. The aqueous phase wasextracted a second time with 500 μL chloroform, this time withoutheptadecanoic acid. The combined organic phases were evaporated at 90°C. After cooling to room temperature, residual fatty acid methyl estersand free fatty acids were dissolved and derivatized in methanolcontaining 0.2 M TMSH (trimethylsulfonium hydroxide).

The regioselectivity of biologically produced (Z)-11-hexadecenoic acidwas determined by comparing the fragmentation patterns of the dimethyldisulfide (DMDS) derivative with the DMDS derivative of an authenticstandard. A yeast culture was split into 12 aliquots (to not change anyparameters in the developed procedure). The cells were pelleted, whichyielded 63 mg cells (ccw) on average (755 mg from 18 mL culture). Thepellets were subjected to base methanolysis as described above. However,after acidification the samples were combined in a 50 mL Falcon tube.The combined sample was extracted two times with 10 mL chloroform. Themixture was centrifuged 10 min at 3000 rpm to achieve a better phaseseparation. The combined organic phases, which were combined in a new 50mL Falcon and were washed consecutively with 10 mL brine and 10 mLwater. The organic phase was dried with anhydrous sodium sulfate andconcentrated in vacuo. The concentrated oil was dissolved in 1.5 mLchloroform and transferred to a 1.5 mL plastic tube. The chloroform wasevaporated at 90° C. The remaining sample was the dissolved in 50 μLmethyl tert-butyl ether (MTBE). The 50 μL were split into 1, 5, 10 and20 μL and transferred into GC-vials without insert. To each vial 200 μLDMDS (dimethyl disulfide) and 50 μL MTBE (containing 60 mg/mL iodine)were added. After the mixture was heated 48 h at 50° C., excess iodinewas removed by the addition of 100 μL saturated sodium thiosulfatesolution. The samples were transferred to plastic vials and extracted totimes with 500 μL dichloromethane. The combined organic phases weretransferred to a new 1.5 mL plastic vial and evaporated at 90° C. Thesamples were taken up in 50 μL DCM and transferred to a GC-vial. Thesample was analyzed by GC-MS (Table 7) using the method of Hagström etal. (2013) (Hagström, A. K. et al. A moth pheromone brewery: productionof (Z)-11-hexadecenol by heterologous co-expression of two biosyntheticgenes from a noctuid moth in a yeast cell factory. Microb. Cell Fact.12: 125 (2013)).

TABLE 7 Analytical parameters used for GC-MS analysis ofDMDS-derivatives System Agilent 6890 N GC, ChemStation G1701EAE02.01.1177 Column Rtx-5 30 m × 320 μm × 25 μm Pressure = 11.74 psi;Flow = 7.1 mL/min Inlet Heater = 250° C.; Pressure = 11.74 psi; TotalFlow {He} = 19.5 mL/min Carrier He @ 147 cm/sec, 11.74 psi Signal Datarate = 2 Hz/0.1 min Oven 80° C. for 2 min Ramp 10° C./min to 180° C.Ramp 3° C./min to 260° C. Ramp 20° C./min to 280° C., hold 10 minInjection Split, 250° C. Split ratio - 1:1 Detector HP 5973 MSD in SCANmode (mass range: 41 to 550 amu) 100 msec Dwell, EMV mode: Gain factor1, 3 min solvent delay, 8.33 cycles/sec Sample Injection volume = 1 uL

Example 5: S. cerevisiae as a Production Platform for Insect FattyAlcohol Synthesis Background and Rationale

Engineering microbial production of insect fatty alcohols from fattyacids requires the functional expression of a synthetic pathway. Onesuch pathway comprises a transmembrane desaturase, and analcohol-forming reductase to mediate the conversion of fatty acyl-CoAinto regio- and stereospecific unsaturated fatty acyl-CoA, andsubsequently into fatty alcohols. A number of genes encoding theseenzymes are found in some insects as well as some microalgae. A numberof gene variants were screened to identify enzyme activities that allowthe creation of pathways capable of high level synthesis of a single ora blend of insect fatty alcohols. Additionally, these enzymes werescreened across multiple hosts (Saccharomyces cerevisiae, Candidatropicalis, and Yarrowia lipolytica) in order to find a suitable hostfor optimum expression of these transmembrane proteins.

Summary of Approach

S. cerevisiae was engineered previously to express select functionaltransmembrane desaturase variants to allow synthesis of(Z)-11-hexadecenoic acid from palmitic acid. This allowed theidentification and rank-ordering of the variants based on theirbioconversion performance (see Example 4).

S. cerevisiae was engineered previously to express select functionaltransmembrane reductase variants to allow synthesis of(Z)-11-hexadecenol (Z11-16OH) from (Z)-11-hexadecenoic acid. Thisallowed the identification and rank-ordering of the variants based ontheir bioconversion performance (see Example 3).

Several fatty alcohol pathways comprised of the most active variantdesaturases and reductases identified in the previous screens wereassembled.

S. cerevisiae W303A and ΔOLE1 were transformed with the pathwayconstructs. Functionality of the pathway was assessed via the ability ofthe recombinant yeasts to synthesize Z11-16OH from palmitic acid.

GC-MS analysis was used to identify and quantify metabolites.

Results

The goal was to engineer one or more insect fatty alcohol biosyntheticpathways in S. cerevisiae. Previously, the functional expression ofseveral transmembrane desaturases of insect origin in S. cerevisiae wasdemonstrated (see Example 4). Briefly, heterologous desaturaseexpression was enabled by designing an expression cassette whichconsists of an OLE1 promoter region, an N-terminal leader sequenceencoding the first 27 amino acids of S. cerevisiae OLE1, and aterminator region of VPS13. Screening for active desaturases was done byusing two approaches. First, active desaturases were screened for theirability to rescue ΔOLE1 growth without exogenous addition of unsaturatedfatty acid (UFA), and second, active desaturases were screened via an invivo screen for bioconversion of palmitic acid into (Z)-11-hexadecenoicacid. These screening strategies allowed the identification of severalactive variants, and the rank ordering of their relative activity. Basedon these screening results, desaturases from Trichoplusia ni (TN_desat)and S. cerevisiae (SC_desat) were selected for combinatorial expressionin fatty alcohol pathways. S. cerevisiae desaturase is known to formpalmitoleic acid and oleic acid.

The functional expression of several transmembrane alcohol formingreductases of insect origin in S. cerevisiae had also been previouslydemonstrated (see Example 3). An expression cassette comprising the GAL1promoter and CYC terminator was used to enable the functional expressionof the reductases in S. cerevisiae. Screening several reductases via invivo bioconversion of (Z)-11-hexadecenoic acid into Z11-16OH allowed theidentification of active variants and rank ordering of their relativeactivity. Based on this screen, reductases from Helicoverpa armigera(HA_reduc), and Spodoptera littoralis (SL_reduc) were chosen forassembly of the fatty alcohol pathways.

Combinatorial assembly created four fatty alcohol pathways, i.e.TN_desat-HA_reduc, TN_desat-SL_reduc, SC_desat-HA_reduc, andSC_desat-SL_reduc. Pathways with SC_desat served as negative control forinsect Z11-16OH synthesis. S. cerevisiae ΔOLE1 and W303A weretransformed with constructs harboring these pathways, and transformantsthat grew on CM-Ura with 2% glucose and coated with palmitoleic acidwere isolated. To test for fatty alcohol production, individual cloneswere inoculated into CM-Ura medium containing 2% glucose, 1% raffinose,2% galactose. 300 mg/L palmitic acid, and 360 mg/L palmitoleic acid wereadded as bioconversion substrates. Bioconversion using palmitic acidwithout palmitoleic was also tested. Post ˜96 h-cultivation in thepresence of palmitic and palmitoleic acid, culture broth analysisrevealed synthesis of Z11-90H as a major C16 alcohol product at ˜0.2mg/L, and ˜0.3 mg/L in cultivation of ΔOLE1 strains harboringSC_desat-HA_reduc, and TN_desat-HA_reduc, respectively (FIG. 15, FIG.16). A minute amount of Z11-16OH was also detected in pathways with T.ni or S. cerevisiae desaturase, and H. armigera reductase. In general,it was expected that in the presence of palmitic acid and palmitoleicacid, Z9-16OH synthesis was more favorable than Z11-16OH synthesisbecause (Z)-11-hexadecenoic acid must be biosynthesized from T. nidesaturase, whereas exogenous addition of palmitoleic acid resulted in amore readily available substrate for synthesis of Z9-16OH. Fatty acidanalysis was also performed. The results showed higher accumulation of(Z)-11-hexadecenoic acid (FIG. 17) in pathways containing insectdesaturase than in pathways expressing S. cerevisiae desaturase. Albeitat minute quantities, detection of Z11-16OH, and (Z)-11-hexadecenoicacid from pathways harboring S. cerevisiae desaturase (which wasunexpected) opens the possibility of a minor All desaturation activityby S. cerevisiae desaturase. Low level synthesis of Z11-16COOH fattyacid moieties can also be derived from elongation of Z9-14COOH fattyacyl intermediate. The data shown in FIG. 15 also showed that incomparison to pathways with H. armigera reductase, the inclusion of S.littoralis reductase resulted in the reduction of (up-to ˜30 fold) inZ9-16OH titer. No Z11-16OH could be detected in pathways employing S.littoralis reductase. These results are consistent with the reductasescreening assay, which showed superior bioconversion of(Z)-11-hexadecenoic acid using H. armigera reductase in comparison to S.littoralis reductase.

The bioconversion of palmitic acid was also tested alone (withoutexogenous addition of palmitoleic acid) by ΔOLE1 strains expressingTN_desat-HA_reduc and TN_desat-SL_reduc (FIG. 18). Culture brothanalysis determined the synthesis of Z11-16OH as the dominantunsaturated C16 fatty acid product (FIG. 16). In this assay, up to 0.22mg/L, and 0.05 mg/L Z11-16OH was synthesized by a pathway harboring H.armigera reductase and S. littoralis reductase, respectively. Thebiologically produced Z11-16OH also matched the retention time andexhibited the characteristic 297.3 m/z peak like the authentic standardZ11-16OH as determined by GC-MS (SIM). Therefore, the regio- andstereoisomer of the biologically produced Z11-16OH was confirmed (FIG.19). Furthermore, Z9-16OH (0.01 mg/L) was also observed in thecultivation of strain co-expressing T. ni desaturase and H. armigerareductase. This suggested that T. ni desaturase may also possess 49desaturation activity.

OLE1 deletion impairs growth. Therefore, pathway expression was alsoexplored in W303A, a host with intact OLE1 allele. However, despitegrowth improvement, pathway expression in this host resulted in morethan two-fold reduction of Z11-16OH titers. This result was likely dueto the repression of OLE1 promoter (which drove heterologous desaturaseexpression) by endogenous unsaturated fatty acyl:CoAs, the products ofOLE1. The S. cerevisiae OLE1 promoter has been previously characterizedwith structural regions found to be positively and negatively regulatedby saturated and unsaturated fatty acid, respectively (Choi, J-Y. et al.Regulatory Elements That Control Transcription Activation andUnsaturated Fatty Acid-mediated Repression of the Saccharomycescerevisiae OLE1 Gene. J. Biol. Chem. 271: 3581-3589 (1996)). In additionto cis-transcriptional regulation, unsaturated fatty acids also interactwith OLE1 promoter elements to regulate mRNA stability (Gonzales, C. I.et al. Fatty acid-responsive control of mRNA stability. Unsaturatedfatty acid-induced degradation of the Saccharomyces OLE1 transcript. J.Biol. Chem. 271: 25801-25809 (1996)). Due to this inherent complexity ofthe OLE1 promoter, the utilization of unregulated orthogonal promoters,such as the OLE1 promoter from S. kluyveri (Kajiwara, S. Molecularcloning and characterization of the v9 fatty acid desaturase gene andits promoter region from Saccharomyces kluyveri. FEMS Yeast. Res. 2:333-339 (2002)) to drive insect desaturase expression can be explored toenhance fatty alcohol production.

In summary, functional expression of synthetic pheromone pathwayvariants in S. cerevisiae ΔOLE1 resulted in the synthesis of Z11-16OHand Z9-16OH from palm oil fatty acids (palmitic acid and palmitoleicacid) up to approximately 0.2 mg/L and 0.3 mg/L, respectively.

The engineered pathway that resulted in the highest fatty alcohols iscomprised of T. ni desaturase and H. armigera reductase.

Accumulation of (Z)-11-hexadecenoic acid, an intermediate of thepathway, was also observed in strains that produced Z11-16OH.

No Z11-16OH was produced and only trace Z9-16OH was detected in thenegative control strain (harboring vector only).

The regio- and stereochemistry of the biologically produced Z11-16OHwere confirmed by comparing the retention time and fragmentation patternto the authentic standard compound via GC-MS.

Conclusions

The engineering of Baker's yeast for synthesis of Z11-16OH and Z9-16OH,fatty alcohol precursors of insect pheromones, was demonstrated.

Fatty alcohol production varies depending on the selection of thedesaturase and reductase variants.

Accumulation of (Z)-11-hexadecenoic acid suggested the possibility offurther fatty alcohol improvement by increasing the performance ofalcohol forming reductase. However, it is also possible that detectionof (Z)-11-hexadecenoic acid was due to its incorporation as phospholipidinto any membrane other than the endoplasmic reticulum membrane (such asmitochondrial membranes, peroxisome, nuclear envelope, etc), thereforeinaccessible to alcohol forming reductase (presumably translocated intothe endoplasmic reticulum) which must utilize (Z)-11-hexadecenoic acidin its CoA thioester moiety as its substrate.

Culture conditions can be explored to increase fatty alcohol titers. TheT. ni desaturase can be replaced in the pathway by A. transitelladesaturase, another variant that also showed high activity and rescuedΔOLE1 growth faster than T. ni desaturase. The synthetic pathway can beimported into Candida tropicalis and Yarrowia lipolytica, which areyeasts with high adhesion property to hydrophobic substrates such aspalmitic and palmitoleic acid. By increasing substrate accessibility tothe microbial production platform, it is foreseeable that product titerand yield can be improved.

Materials & Methods Strain Construction and Functional Expression Assay

S. cerevisiae ΔOLE1 (MATA OLE1::LEU2 ura3-52 his4), and W303A (MATAura3-1 trp1-1 leu2-3_112 his3-11_15 ade2-1 can1-100) were used asexpression hosts. Modular design allows combinatorial pathway assemblyutilizing BamHI and XhoI to excise reductase synthons (see Example 3)and subcloning into plasmids containing pOLE1-desaturase constructs (seeExample 4). Competent yeasts were transformed with pathway constructsand plated on CM-Ura glucose agar plate (Teknova). In the case of ΔOLE1transformation, colony plating utilized 20 mM CM-Ura glucose agar platesthat were coated with 100 μL CM-Ura glucose medium containing 1%tergitol and 3 μL palmitoleic acid.

To assess functional expression, transformants were inoculated in ˜20 mLCM-Ura liquid medium containing 6.7 g/L of YNB, 2% glucose, 1%raffinose, and 2% galactose. Fatty acid substrates, i.e. palmitic acid(in ethanol), was added at a final concentration of 300 mg/L.Palmitoleic acid was added at a final concentration of 360 mg/L.Bioconversion assay proceeded for 96 h at 28° C. prior to GC-MSanalysis.

Metabolite Extraction and GC-MS Detection

Fatty acid analysis was as described in Example 4, except that insteadof extracting the sample two times, the sample was only extracted oncewith chloroform containing 1 mM methyl heptadecanoate (C17:0Me). Fattyalcohol analysis was as described in Example 3, except that instead ofhexane (containing tetradecanedioic acid), chloroform (containing 1 mMmethyl heptadecanoate) was used. The extraction time was reduced from 1h to 20 s. Afterwards the samples were collected in a 1.8 mL GC vial andnot in a 1.5 mL plastic tube. The mass spectrometer was used in SIM mode(m/z 208, 297.3 and 387.3).

Example 6: Expression of Transmembrane Desaturases in Candida tropicalisBackground and Rationale

Engineering microbial production of insect fatty alcohols from fattyacids requires the functional expression of a synthetic pathway. Onesuch pathway comprises a transmembrane desaturase, and analcohol-forming reductase to mediate the conversion of fatty acyl-CoAinto regio- and stereospecific unsaturated fatty acyl-CoA, andsubsequently into fatty alcohols. A number of genes encoding theseenzymes are found in some insects as well as some microalgae. A numberof gene variants were screened to identify enzyme activities that allowthe creation of pathways capable of high level synthesis of a single ora blend of insect fatty alcohols. Additionally, these enzymes can bescreened across multiple hosts (Saccharomyces cerevisiae, Candidatropicalis, and Yarrowia lipolytica) to optimize the search towardfinding a suitable host for optimum expression of these transmembraneproteins.

Summary of Approach

A small set of desaturases (insect origin: Agrotis segetum, Amyeloistransitella, Helicoverpa zea, Trichoplusia ni, Ostrinia furnacalis, andLampronia capitella and marine diatom: Thalassiosira pseudonana) wereselected as a test case to explore and establish functional expressionassays, metabolite extraction methods, and analytical chemistry.

Successful integration and functional expression of mCherry control frompXICL expression cassette in SPV053 were confirmed.

A recombinant desaturase library using the same pXICL vector in SPV053background was integrated (FIG. 20). One variant, the Z11 desaturase ofAgrotis segetum, was also cloned to produce a protein product with thefirst 27 amino acids of Candida albicans Ole1p fused to the N-terminusof the insect desaturase (SEQ ID NO: 15).

Functionality of the desaturase was validated via an in vivobioconversion of hexadecanoic acid (palmitic acid) into(Z)-11-hexadecenoic acid (palmitvaccenic acid).

GC-FID and GC-MS analyses were used to identify and quantifymetabolites.

Results Library Construction

This study focused on the screening for transmembrane desaturasevariants in C. tropicalis (SPV053). Five insect desaturases withreported Z11 desaturase activity on palmitoyl-CoA (C16:0) (SEQ ID NOs:16-19, 23) and three insect desaturases with reported Z9 desaturaseactivity (SEQ ID NOs: 20-22) were included in the screen. One variant,the Z11 desaturase from A. segetum (SEQ ID NO: 16), was also cloned with27 amino acids of the Candida albicans OLE1 N-terminus fused upstream ofthe insect sequence (FIG. 20, SEQ ID NO: 15). At the time ofconstruction, the A. segetum Z11 desaturase was believed to be apositive control and the C. albicans OLE1 fusion was constructed to testif inclusion of a Candida leader sequence would improve functionalexpression. The construct was designed to mimic those used inSaccharomyces cerevisiae desaturase screening (See Example 4). Finally,a control construct expressing mCherry red fluorescent protein (SEQ IDNO: 14) was included to act as a positive control for integration andexpression and a negative control for recombinant desaturase activity(FIG. 21A-FIG. 21D).

Transformation efficiencies of linearized plasmids into SPV053 variedgreatly across constructs. Despite low efficiencies, at least 3 clonalisolates were identified for each variant (Tables 8 and 9). It had beenhypothesized that larger colonies on transformation plates were morelikely to be positive integrants because the presence of the Zeocinresistance marker should increase growth rate under Zeocin selection.Analysis of the screening results suggested that the number of largecolonies is not correlated to transformation efficiency. Instead totalcolony (small and large) count correlated best with observed efficiency(FIG. 22). In addition, in some cases positive clones were found amongthe small colonies. It is possible that at lower plating density growthrate may be correlated with integration events (i.e. positive integrantsgrow faster). A secondary screen of repatching colonies on YPD+Zeocinproved effective in enriching for positive integrants. Fast growingpatches were more likely to be positive integrants than the generalpopulation of colonies on transformation plates.

TABLE 8 Desaturase transformations in SPV053. Efficiency oftransformation varied across constructs with a relatively high degree ofbackground under Zeocin selection. large colonies total colonies speci-pXICL DNA (control (control ficity source species plasmid ug plate)plate) con- mCherry_Ct pPV0137 1.1  60 (30)  2,000 (600) trol Z11Argotis segetum- pPV0138 1.2 120 (78) >10,000 (320) OLE1_Ca Agrotissegetum pPV0139 1.3 115 (78)  8,000 (320) Amyelois transitella pPV01401.1 220 (78)  5,000 (320) Trichoplusia ni pPV0141 1.1 100 (78) >10,000(320) Helicoverpa zea pPV0142 1.0 350 (78)  5,000 (320) ThalassiosirapPV0146 1.1 140 (78)  1,500 (320) pseudonana Z9 Ostrina FurnacalispPV0143 0.9 220 (78)  6,000 (320) Lampronia capitella pPV0144 1.2 230(78)  5,000 (320) Helicoverpa zea pPV0145 1.2  72 (78)  2,000 (320)

TABLE 9 Desaturase SPV053 library construction. Five insect desaturaseswith putative Z11 desaturation activity and 3 insect desaturases withputative Z9 desaturation activity were integrated into the SPV053background using the pXICL vector. In addition, a control strainexpressing mCherry was constructed with the same vector. pXICL TotalTotal Fraction specificity source species plasmid positives screenedpositive control mCherry_Ct pPV0137 16 16 1.00 Z11 Argotis segetum-pPV0138 7 12 0.58 OLE1_Ca Agrotis segetum pPV0139 12 12 1.00 Amyeloistransitella pPV0140 5 60 0.08 Trichoplusia ni pPV0141 6 12 0.50Helicoverpa zea pPV0142 5 120 0.04 Thalassiosira pPV0146 3 96 0.03pseudonana Z9 Ostrina Furnacalis pPV0143 3 57 0.05 Lampronia capitellapPV0144 3 58 0.05 Helicoverpa zea pPV0145 3 94 0.03

Functional Expression Assay

Functional expression of the heterologous desaturases was characterizedby a series of in vivo bioconversion experiments. C. tropicalis SPV053derived stains expressing insect desaturases were cultured in rich (YPD)or defined (CM glucose) media supplemented with ethanol (for induction)and saturated acid substrates (palmitic acid, methyl palmitate, methylmyristate). Small scale (2 ml) cultures were cultivated for a total of72 hours in 24 deep well plates with substrate added after the initial24 hours.

The first screen examined multiple bioconversion media withsupplementation of a palmitic acid substrate. Two functionalpalmitoyl-CoA (Z)-11 desaturases were identified by fatty acid methylester (FAME) analysis of the cellular lipid content. Strains expressingA. transitella or H. zea Z11 desaturases (SPV0305-SPV0310) produced afatty acid species not observed in the mCherry control strains(SPV0302-SPV0304) which eluted with the (Z)-11-hexadecenoic acidstandard (FIG. 23). No other tested strains produced non-native fattyacid species (data not shown). Approximate fatty acid composition of theC16-fraction is listed in Table 10. The native palmitoyl-CoA (Z)-9desaturase is still present in the SPV053 background which means the(Z)-9/(Z)-11 specificity of the desaturases cannot be rigorouslydetermined. Supplementation of palmitic acid in the media increased the(Z)-11/(Z)-9 hexadecenoic acid ratio from 0.6 to 1.4 for H. zeadesaturase expressing strains. (Z)-11-hexadecenoic acid titers wereobserved to be approximately 5.62 mg/L for strains expressing A.transitella desaturase and 5.96 mg/L for strains expressing H. zeadesaturase. Similar performance was observed with methyl palmitatesupplementation (data not shown).

TABLE 10 Composition of the C16-fatty acid fraction in different C.tropicalis SPV053 expressing different desaturases. *NS = no substrate(hexadecanoic acid) was added. C16:0 Z9-C16:1 Z11-C16:1 Z11/Z9 [%] [%][%] ratio mCherry 72.9 27.1 0.0 0.0 Hzea-YPD_NS 50.0 30.9 19.1 0.6Hzea-YPD 58.1 17.5 24.4 1.4 AT-YPD 55.5 14.5 30.0 2.1

The bioconversion assay was scaled-up to 20 ml in shake flasks in orderto generate enough biomass for additional characterization of theputative (Z)-11-hexadecenoic acid species. While the observed specieseluted with the (Z)-11-hexadecenoic acid standard and independently ofthe (Z)-9-hexadecenoic acid standard, it was possible that a differentfatty acid isomer (e.g. (E)-9-hexadecenoic acid) could have a similarretention time to (Z)-11-hexadecenoic acid. As different stereoisomerselute differently on the DB-23 the occurrence of (E)-11-hexadecenoiccould be excluded. Final confirmation of (Z)-11-hexadecenoic acidproduction was completed by using mass spectroscopy detection of DMDSderivatized fatty acids to confirm the 11-regioselectivity. Using thisderivatization technique (Z)-11 and (E)-11 isomers could in principlealso be resolved. The fragmentation pattern of experimental samplescould be matched to the (Z)-11-hexadecenoic acid standard (FIG.24A-24E). Using this technique, production of the specific(Z)-11-hexadecenoic acid regio- and stereoisomer was confirmed for bothA. transitella and H. zea desaturase expressing strains.

Finally, methyl myristate (C14:0) was tested as substrate for the entiredesaturase library. A non-native fatty acid species which elutes betweenmyristate (C14:0) and (Z)-9-tetradecenoic acid (Z9-C14:1) was observedin strains expressing either A. transitella or H. zea Z11 desaturases(FIG. 25A). It is hypothesized that this non-native species is(Z)-11-tetradecenoic acid, and this can be confirmed with an authenticstandard. In addition, A. segetum Z11 desaturase, O. furnacalis Z9desaturase, and H. zea Z9 desaturase all produced a shoulder peak whicheluted just after the myristate (C14:0) peak (FIG. 25B). Other C14derived species (e.g. tetradecanedioic acid) were observed in allstrains. These results suggest that A. transitella and H. zeadesaturases have some activity on myristoyl-CoA. Confirmation of unknownspecies and quantification is required to draw further conclusions aboutdesaturase substrate specificity in vivo.

In summary, two desaturases from Helicoverpa zea (AAF81787, SEQ ID NO:39) and from Amyelois transitella (JX964774), were expressed in SPV053and conferred synthesis of (Z)-11-hexadecenoic acid from eitherendogenously produced or supplemented palmitic acid.

Functional expression of H. zea and A. transitella desaturases in C.tropicalis SPV053 was confirmed using an in vivo bioconversion assay inboth rich (YPD) and defined (CM glucose) media. The active desaturasesgenerated intracellular (Z)-11-hexadecenoic acid which was not observedin mCherry expressing control strains. C16-fatty acid composition ofSPV053 expressing H. zea desaturase is approximately 50.0% hexadecanoicacid, 30.91% (Z)-9-hexadecenoic acid and 19.1% (Z)-11-hexadeceneoicacid. With palmitic acid supplementation the composition is 58.1%hexadecanoic acid, 17.5% (Z)-9-hexadecenoic acid and 24.4%(Z)-11-hexadeceneoic acid. The C16-fatty acid composition of SPV053expressing A. transitella desaturase is 55.5% hexadecanoic acid, 14.5%(Z)-9-hexadecenoic acid and 30.0% (Z)-11-hexadeceneoic acid. Incomparison, SPV053 expressing mCherry produced a C16-fatty acidcomposition of approximately 72.9% hexadecanoic acid, 27.1%(Z)-9-hexadecenoic acid and no (Z)-11-hexadeceneoic acid.(Z)-11-hexadecenoic acid was produced at approximately 5.5 mg/L in bothstrains expressing functional Z11 desaturases.

No (Z)-11-hexadecenoic acid was observed in strains harboring T. ni, Tpseudonana, or A. segetum desaturase.

No difference in fatty acid composition was observed for strainsexpressing Z9 insect desaturases from H. zea, O. furnacalis, or L.capitella.

The regio- and stereoisomer of the biologically produced(Z)-11-hexadecenoic acid were confirmed by comparing the retention timeand fragmentation pattern of the authentic standard compound via GC-MSafter DMDS derivatization.

Bioconversions of SPV053 expressing A. transitella and H. zeadesaturases with supplementation of methyl myristate produced anunidentified metabolite not observed in the mCherry expressing negativecontrol strain. The GC retention time of this metabolite is foundbetween myristate (C14:0) and (Z)-9-tetradecenoic acid.

Conclusions

Functional expression of transmembrane desaturase of insect origin in C.tropicalis SPV053 has been achieved.

The active desaturases identified via screening in C. tropicalis alsocomplemented OLE1 function when expressed in S. cerevisiae ΔOLE1 (SeeExample 4).

An in vivo assay can be used to assay desaturase activity in C.tropicalis for non-native fatty acid isomers (e.g. (Z)-11-hexadecenoicacid). Enhanced ratios of non-native fatty acids can be produced withsupplementation of saturated acid substrates such as palmitic acid ormethyl myristate.

Functional expression and/or activity of insect desaturases varieswidely in C. tropicalis SPV053 depending on sequence origin. Similar toresults observed in the S. cerevisiae screen (See Example 4), A. segetumand T. pseudonana variants did not produce detectable(Z)-11-hexadecenoic acid. Interestingly, T. ni desaturase also failed toproduce detectable (Z)-11-hexadecenoic acid under assay conditions.Unlike in the S. cerevisiae assay, the T. ni expression construct didnot include a chimeric OLE1 leader sequence.

The inclusion of the C. albicans OLE1 leader sequence on the functionalH. zea variant and non-functional T. ni variant can be tested.

The functional expression of additional desaturase variants to identifyC14-specific desaturases can be explored.

Expression of functional desaturase with reductase variants can be doneand subsequent screen for unsaturated fatty alcohol production can beperformed.

Materials & Methods Strain Construction

A conservative approach was used for recoding of genes. Native sequenceswere unaltered except for replacement of CTG leucine codons with TTA.All genes were cloned into pPV0053 using NcoI and NotI restriction sitesby Genscript. After transformation into E. coli NEB10β, plasmids wereminiprepped using the Zyppy Plasmid Miniprep Kit (Zymo Research, Irvine,Calif.). Plasmids were linearized by digestion with BsiWI (New EnglandBiolabs, Ipswich, Mass.) before transformation into SPV053. Afterdigestion, DNA was isolated using Clean and Concentrator Kit (ZymoResearch, Irvine, Calif.). Approximately 1 μg of DNA was transformed byelectroporation. Instead of incubation with TE+100 mM lithiumacetate+DTT, cells were incubated in only TE+100 mM lithium acetate for2 hours. Positive integrants were found to be site-specific andgenotyping was conducted by check PCR. A two-stage approach was adoptedfor further screening of low efficiency transformations. Approximately60 colonies were re-patched on YPD+300 μg/ml Zeocin and grown overnight.The subset of patches which grew quickly (dense growth within 24 hours)were screened by colony PCR. The vast majority of rapid growing patcheswere identified as positive integrants.

Functional Expression Assay

Palmitic Acid Supplementation in YPD and CM Glucose

Positive isolates were re-patched onto YPD+300 μg/ml Zeocin and grownovernight and then stored at 4° C. Strains were inoculated from patchplates into 2 ml of YPD in 24 deep well plates (square well, pyramidbottom). Three positive clones were inoculated for each desaturasevariant and the mCherry expressing control strain. Deep well plates wereincubated at 30° C., 1000 rpm, and 80% humidity in the Infors HTMultitron Pro plate shaker for 24 hrs. After 24 hrs of incubation,cultures were split into equal 1 ml volumes to make two sets ofidentical plates. Both sets of plates were pelleted by centrifugation at500×g. One set of plates was resuspended in 2 ml of YPD+0.3% (v/v)ethanol and the second set was resuspended in 2 ml of CM glucose+0.3%ethanol. Ethanol was added at this stage to induce recombinant enzymeexpression from the ICL promoter. Cultures were incubated for another 24hours under the same conditions before 300 mg/L palmitic acid was addedto cultures from a 90 g/L stock solution in ethanol. The result was theaddition of a fresh 0.3% ethanol in conjunction with the palmitic acid.A subset of strains was also cultured without palmitic acid addition.These cultures had 0.3% ethanol added instead. All cultures wereincubated for an additional 24 hrs before a final addition of 0.3%ethanol. After another 24 hr period of incubation, 1.5 ml of eachculture was harvested in 1.7 ml microcentrifuge tubes and pelleted.Supernatant was saved in fresh tubes and pellets were processed asdescribed below. A subset of supernatant samples was also extracted tolook for free acid in the extracellular medium.

Repeated Screening with Alternate Substrates

The mCherry control and confirmed positive variants were rescreenedusing both palmitic acid and methyl palmitate as substrates. Theculturing was conducted as described above with equimolar (1.17 mM)amounts of substrate added from ethanol stock solutions (methylpalmitate 94 g/L stock, 313 mg/L final concentration). The same protocolwas also repeated with the full panel of strains using an 84 g/L stockof methyl myristate (C14:0). The final concentration of substrate wasagain 1.17 mM.

Confirmation of (Z)-11-Hexadecenoic Acid Isomer

The in vivo bioconversion assay was scaled up for confirmation of(Z)-11-hexadecenoic acid synthesis. 2 ml YPD seed cultures of strainsSPV0302, SPV0303, and SPV0304 (mCherry), SPV0304, SPV0305, and SPV0306(A. transitella Z11 desaturase), and SPV0307, SPV0308, and SPV0309 (H.zea Z11 desaturase) were grown overnight at 30° C., 1000 rpm, 80%humidity in the Infors HT Multitron plate shaker. 200 μl of overnightculture from each of the three clonal isolates was pooled and inoculatedinto a single 125 ml baffled flask containing 20 ml YPD. The resultingthree flasks were grown for 24 hrs at 30° C. and 250 rpm (Infors Flaskshaker). Cultures were pelleted by centrifugation at 500×g andresuspended in 20 ml of YPD+0.3% (v/v) ethanol and returned to 125 mlbaffled shake flasks. Cultures were incubated for an additional 24 hoursbefore addition of 300 mg/L palmitic acid in a 90 g/L stock in ethanol(221 μl per flask). After 24 hours of incubation another 0.3% (v/v)ethanol (221 μl) was added to each flask for sustained induction. Flaskswere incubated for an additional 24 hours before cells were harvestedfor FAME analysis and DMDS derivatization.

Metabolite Extraction and GC-MS Detection

Total lipid composition as well as the (Z)-11-hexadecenoic acidquantification was based on modified procedures by Moss et al. (1982)and Yousuf et al (2010). The pelleted cells (in 1.5 mL plastic tubes),usually about 10 mg to 80 mg, were resuspended in methanol containing 5%(w/w) of sodium hydroxide. The alkaline cell suspension was transferredinto a 1.8 mL screw-cap GC-vial. The mixture was heated for 1 h in theheat block at 90° C. Prior to acidification with 400 2.5 N HCl the vialwas allowed to cool to room temperature. 500 μL chloroform containing 1mM heptadecanoic were added and the mixture was shaken vigorously, thenboth aqueous and organic phase were transferred into a 1.5 mL plastictube. The mixture was centrifuged at 13,000 rpm, afterwards 450 μL ofthe organic phase were transferred into a new 1.5 mL plastic tube. Theaqueous phase was extracted a second time with 500 μL chloroform, thistime without heptadecanoic acid. The combined organic phases wereevaporated at 90° C. After cooling to room temperature, residual fattyacid methyl esters and free fatty acids were dissolved and derivatizedin methanol containing 0.2 M TMSH (trimethylsulfonium hydroxide).

The regioselectivity of biologically produced (Z)-11-hexadecenoic acidwas determined by comparing the fragmentation patterns of the dimethyldisulfide (DMDS) derivative with the DMDS derivative of an authenticstandard. A yeast culture was split into 12 aliquots (to not change anyparameters in the developed procedure). The cells were pelleted, whichyielded 63 mg cells (ccw) on average (755 mg from 18 mL culture). Thepellets were subjected to base methanolysis as described above. However,after acidification the samples were combined in a 50 mL falcon tube.The combined sample was extracted two times with 10 mL chloroform. Themixture was centrifuged 10 min at 3000 rpm to achieve a better phaseseparation. The combined organic phases were combined in a new 50 mLfalcon and were washed consecutively with 10 mL brine and 10 mL water.The organic phase was dried with anhydrous sodium sulfate andconcentrated in vacuo. The concentrated oil was dissolved in 1.5 mLchloroform and transferred to a 1.5 mL plastic tube. The chloroform wasevaporated at 90° C. The remaining sample was the dissolved in 50 μLmethyl tert-butyl ether (MTBE). The 50 μL were split into 1, 5, 10 and20 μL and transferred into GC-vials without insert. To each vial 200 μLDMDS (dimethyl disulfide) and 50 μL MTBE (containing 60 mg/mL iodine)were added. After the mixture was heated 48 h at 50° C., excess iodinewas removed by the addition of 100 μL saturated sodium thiosulfatesolution; however, due to excessive formation of detergents from theCandida strain, the layer did not mix properly. The samples weretherefore diluted in a 15 mL falcon tube to a final sample compositionof 200 4, 3.55 mL MTBE (containing iodine and analyte), 500 μLdichloromethane, 1.5 mL water and 1 mL ethanol. The organic phase wasevaporated stepwise at 85° C. in a 1.8 mL glass vial. The samples weretaken up in 500 μL dichloromethane and the sample was analyzed by GC-MSusing the method of Hagström et al. (2013) as in Example 4.

Example 7: Expression of Transmembrane Desaturases in Yarrowialipolytica Background and Rationale

Engineering microbial production of insect fatty alcohols from fattyacids requires the functional expression of a synthetic pathway. Onesuch pathway comprises a transmembrane desaturase, and analcohol-forming reductase to mediate the conversion of fatty acyl-CoAinto regio- and stereospecific unsaturated fatty acyl-CoA, andsubsequently into fatty alcohols. A number of genes encoding theseenzymes are found in some insects as well as some microalgae.Alternatively, regio- and stereospecific desaturases can be used toproduce a microbial oil rich in fatty acid precursors. The microbial oilcan then be derivatized and reduced to active ingredients. A number ofgene variants were screened to identify enzyme activities that allow thecreation of pathways capable of high level synthesis of a single or ablend of insect fatty acids and alcohols. Additionally, these enzymeswere screened across multiple hosts (Saccharomyces cerevisiae, Candidaviswanathii (tropicalis), and Yarrowia lipolytica) to optimize thesearch toward finding a suitable host for optimum expression of thesetransmembrane proteins.

Initial screening of desaturases in S. cerevisiae and C. viswanathii(tropicalis) identified three active Z11-C16:1 desaturase variants fromAmyelois transitella, Helicoverpa zea, and Trichoplusia ni. The S.cerevisiae screening used coding sequences with an N-terminal leadersequence of the S. cerevisiae Ole1p Z9 desaturase fused to the fulllength insect Z11 desaturase sequence. This strategy has been usedpreviously in the scientific literature to express eukaryoticdesaturases in S. cerevisiae. All three of the above desaturasesdisplayed Z11 desaturase activity with the Ole1p leader fusion whenexpressed in a OLE1 deletion background. An analogous design with a C.albicans Ole1p leader sequence was used with the Z11 desaturase from H.zea. While active, this Ole1p-H. zea desaturase fusion did notsignificantly increase Z11-hexadecenoic acid titer. Additionally, aconservatively optimized A. transitella Z11 desaturase was active inboth S. cerevisiae and C. viswanathii. The following study focused ontesting the functional expression of the H. zea, T. ni, and A.transitella Z11 desaturases in two different Y. lipolytica strains,SPV140 and SPV300. Both native and Homo sapiens codon optimizedsequences were used for the H. zea and T. ni desaturases while only thenative sequence was used for A. transitella. Finally, the N-terminus ofthe Y. lipolytica Ole1p Z9 stearoly-CoA desaturase aligns more closelywith insect desaturases than the N-terminus of Ole1p from either S.cerevisiae or C. albicans. Based on this alignment two additionaldesaturase versions were created. A putative leader sequence was swappedfrom the Y. lipolytica Ole1p onto the T. ni and H. zea desaturases.

Summary of Approach

A focused library of Z11 desaturases (insect origin: Amyeloistransitella, Helicoverpa zea, Trichoplusia ni), which had observedactivity in either S. cerevisiae or C. viswanathii were cloned into adouble crossover cassette targeting the XPR2 locus with a URA3 selectionmarker. Protein coding sequences use either the native insect sequence(SEQ ID NOs: 24, 25), Homo sapiens optimized coding sequence (SEQ IDNOs: 26, 27), or the Homo sapiens optimized sequence with the N-terminal84 bases (H. zea, SEQ ID NO: 29) or 81 bases (T. ni, SEQ ID NO: 28)swapped for the N-terminal 96 bases of the Y. lipolytica OLE1(YALI0005951) gene. Unlike in the S. cerevisiae and C. viswanathiiscreens, the leader sequence chimeras test a direct swap of leadersequences instead of concatenating a host leader sequence to theN-terminus of the full length desaturase coding sequence. Only thenative coding sequence was used for the A. transitella desaturase (SEQID NO: 30).

Each of the 7 desaturase constructs was transformed into SPV140 (PO1f)and SPV300 (H222 ΔP ΔA ΔF ΔURA3) and site-specific integrants wereconfirmed.

Desaturase activity was tested via an in vivo bioconversion ofhexadecanoic acid (palmitic acid) into (Z)-11-hexadecenoic acid(palmitvaccenic acid) in YPD medium.

GC-FID analyses were used to identify and quantify metabolites.

Results Strain Construction

Desaturase variants were cloned into the pPV101 vector which contains aY. lipolytica expression cassette targeting integration into the XPR2locus.

The T. ni and H. zea desaturases were each synthesized with the nativeinsect sequence (SEQ ID NOs: 24, 25), full length insect sequence codonoptimized for Homo sapiens (SEQ ID NOs: 26, 27), or with the putativeleader sequence replaced by the leader sequence from Y. lipolytica OLE1desaturase (SEQ ID NOs: 28, 29). The A. transitella desaturase was alsosynthesized using the native insect coding sequence (SEQ ID NO: 30). Allseven desaturase variants were transformed into SPV140. Based onprevious activity results, only the H. zea and A. transitella desaturasevariants were transformed into SPV300.

Functional Expression Assay

Functional activity was assessed by a modification of the protocol usedfor transmembrane desaturase expression in C. viswanathii SPV053 (SeeExample 6). Briefly, Y. lipolytica SPV140 and SPV300 derived stainsexpressing insect desaturases were cultured in rich (YPD) to generatebiomass. Using the YPD generated biomass, small scale (2 ml) cultureswere cultivated with palmitic acid for a total of 48 hours in 24 deepwell plates (See Materials & Methods for detail).

In the initial screen of T. ni, H. zea, and A. transitella variants,only H. zea desaturase variants that were codon optimized for Homosapiens produced detectable Z11-hexadecenoic acid (FIG. 26). Expressionof native H. zea desaturase conferred production of 100±5 mg/LZ11-hexadecenoic acid and the version with a Y. lipolytica OLE1 leadersequence produced 83±11 mg/L. As seen in FIG. 26, the distribution ofthe other major fatty acid species was relatively unaffected byfunctional desaturase expression. In the active strains,Z11-hexadecenoic acid made up ˜10% (g/g) of the fatty acid species(including palmitic acid substrate which may be adsorbed to the outercell surface).

A follow up experiment was conducted comparing active variants in theSPV140 background to SPV300 derived desaturase strains. The parentSPV300 and SPV140 expressing hrGFP were used as negative controls. Thesame bioconversion assay protocol was used. As in SPV140, only H.sapiens optimized variants produced detectable activity (FIG. 27).SPV300 strains grew to higher final cell densities (SPV300 OD600=26-28,SPV140 OD600=19-22) (FIG. 28). The highest titers were observed forstrains expressing the native H. zea Z11-desaturase with H. sapienscodon optimization (pPV199). The retested SPV140 strains produced 113±1mg/L (5.5±0.2 mg/L/OD) Z11-hexadecenoic acid which is 13% higher thantiters observed in the first experiment (FIG. 29). SPV300 strainsexpressing the same desaturase generated a wider range of productivity.On average they produced 89±18 mg/L (3.3±1.2 mg/L/OD) Z11-hexadecenoicacid, but one clone produced 124 mg/L (4.6 mg/L/OD) Z11-hexadecenoicacid.

In summary, only the H. zea Z11 desaturase variants with Homo sapienscodon optimization produced detectable Z11-hexadecenoic acid. Under thecurrent assay condition, marginally higher titers were observed in theSPV140 background over SPV300. Table 11 summarizes the Z11-hexadecenoicacid titers.

TABLE 11 Z11-hexadecenoic acid titers obtained from expression ofexemplary desaturases in Yarrowia lipolytica Codon Z11-hexadecenoicDesaturase optimization Parent Strain acid titer (mg/L) Z11 T. ni NativeSPV140 ND (no detection) Z11 T. ni Homo sapiens SPV140 ND Yl OLE1-Z11 T.ni Homo sapiens SPV140 ND Z 11 H. zea Native SPV140 ND SPV300 ND Z11 H.zea Homo sapiens SPV140 100 ± 5  SPV300 87 ± 18 Yl OLE1-Z11 H. zea Homosapiens SPV140 83 ± 11 SPV300 55 ± 1  Z11 A. transitella Native SPV140ND SPV300 ND

In SPV300, one non-site-specific integrant of pPV200 (Y. lipolyticaOLE1-H. zea Z11 desaturase with Homo sapiens codon optimization) wastested. This integrant did not produce detectable Z11-hexadecenoic acid,while the two site-specific integrants produced 55±1 mg/L.

No major hydroxy or diacid peaks were observed from pellets of SPV140 orSPV300 derived strains, and deletion of β-oxidation/co-oxidation genesin SPV300 did not increase Z11-hexadecenoic acid accumulation under thecurrent assay condition (relatively low substrate concentration, richmedium).

Conclusions

The H. zea Z11 desaturase is active and confers production of ˜100 mg/LZ11-hexadecenoic acid, from ˜500 mg/L palmitic acid substrate. Thefunctional expression was demonstrated across three positive integrantsand replicate experiments in a 24 well plate assay.

H. zea desaturase required codon optimization (Homo sapiens orpotentially Y. lipolytica) for activity in Y. lipolytica.

The T. ni Z11 desaturase, while active in S. cerevisiae, does notproduce detectable Z11-hexadecenoic acid in Y. lipolytica.

The reproducibility of the assay for Y. lipolytica strains can beconfirmed starting from glycerol stock.

A. transitella desaturase can be codon optimized for expression in Y.lipolytica.

Since Y. lipolytica is a candidate production host, additional copies ofactive desaturases can be integrated in Y. lipolytica, cultureconditions to improve bioconversion can be identified, and substrateconversion can be quantified.

Materials & Methods Strain Construction

All desaturase genes were synthesized (Genscript). Either nativesequences or Homo sapiens codon optimization was used. Synthesized geneswere subcloned into pPV101. Plasmids were transformed and prepped fromE. coli EPI400 using the Zyppy Plasmid Miniprep Kit (Zymo Research,Irvine, Calif.). Approximately ˜1-2 μg of linearized DNA was transformedusing Frozen-EZ Yeast Transformation II Kit (Zymo Research, Irvine,Calif.). The entire transformation mixture was plated on CM glucose -uraagar plates. Positive integrants were found to be site-specific andgenotyping was conducted by check PCR.

Functional Expression Assay

Palmitic Acid Supplementation in YPD

Positive isolates were re-patched onto YPD, grown overnight, and thenstored at 4° C. Strains were inoculated from patch plates into 2 ml ofYPD in 24 deep well plates (square well, pyramid bottom). Three positiveclones were inoculated for each desaturase variant. Three isolates ofpPV101 in SPV140 and the parent SPV300 were used as negative controls.Deep well plates were incubated at 28° C. and 250 rpm in the InforsMultitron refrigerated flask shaker for 24 hrs. After 24 hrs ofincubation, a 1 ml volume of each culture was pelleted by centrifugationat 500×g. Each pellet was resuspended in 2 ml of YPD. 500 mg/L palmiticacid was added to cultures from a 90 g/L stock solution in ethanol. Theresult was the addition of 0.5% ethanol with the palmitic acidsubstrate. All cultures were incubated for 48 hours before endpointsampling. Final cell densities were measured with the Tecan Infinite200pro plate reader. 0.75 or 0.8 ml of each culture was harvested in 1.7ml microcentrifuge tubes and pelleted. Supernatant was removed andpellets were processed as described below.

Metabolite Extraction and GC-FID Analysis

Total lipid composition as well as the (Z)-11-hexadecenoic acidquantification was based on modified procedures by Moss et al. (1982)and Yousuf et al (2010). The pelleted cells (in 1.5 mL plastic tubes),usually about 10 mg to 80 mg, were resuspended in methanol containing 5%(w/w) of sodium hydroxide. The alkaline cell suspension was transferredinto a 1.8 mL crimp vial. The mixture was heated for 1 h in the heatblock at 90° C. Prior to acidification with 400 2.5 N HCl the vial wasallowed to cool to room temperature. 500 μL chloroform containing 1 mMmethyl heptadecanoate were added and the mixture was shaken vigorously,then both aqueous and organic phase were transferred into a 1.5 mLplastic tube. The mixture was centrifuged at 13,000 rpm, afterwards 450μL of the organic phase were transferred into a GC vial. For theanalysis of lipids and the quantification of fatty acids 50 μL of 0.2 MTMSH (trimethylsulfonium hydroxide in methanol) was added and the sampleanalyzed by GC-FID.

Example 8: Candida viswanathii (tropicalis) as a Production Platform forInsect Fatty Alcohol Synthesis Background and Rationale

Variants of insect transmembrane desaturases and reductases werepreviously screened and rank-ordered based on their functionalexpression in either Candida viswanathii or Saccharomyces cerevisiae(see Examples 3, 4 and 6). Helicoverpa zea desaturase and Helicoverpaarmigera reductase were selected to assemble a synthetic insect fattyalcohol pathway in C. viswanathii. Simultaneous expression of codonoptimized H. zea desaturase under Candida isocitrate lyase (ICL)promoter, and codon optimized H. armigera reductase under Candidatranscription elongation factor (TEF) promoter was achieved via genomicintegration of the full fatty alcohol pathway. Accumulation of Z11-16OHwas achieved in cultures of the recombinant strain (SPV0490) usingsimple carbon sources and palmitic acid.

Summary of Approach

Integration plasmids were designed containing a functional Helicoverpazea desaturase (See Example 6) paired with a Helicoverpa armigerareductase driven by a putatively constitutive C. tropicalis promoter(pTEF).

Functionality of the full pathway was assessed via an in vivobioconversion of hexadecanoic acid (palmitic acid) into Z11-16OH.

GC-FID and GC-MS analyses were used to identify and quantifymetabolites.

Results

Accumulation of Z11-16OH was detected in cultures of Candida engineeredto express H. zea desaturase under an ICL promoter and H. armigerareductase under a TEF promoter (Table 12 and FIG. 30).

TABLE 12 Tabulated Z11-16OH titers from Candida viswanathiibioconversion assay. SPV088 is C. viswanathii which was engineered toexpress mCherry (negative control). SPV0490 is C. viswanathii which wasengineered to express the insect fatty alcohol pathway. Z11-16OH titers(mg/L) SPV0488 (negative control) SPV0490 Sample 1 0.08 1.03 Sample 20.07 0.93 Sample 3 0.06 0.88 Average 0.07 0.95 StDev 0.01 0.06

Materials & Methods Strain Construction

The integration plasmid (ppV0228) was designed to contain two expressioncassettes. The first cassette contains H. zea codon-optimized desaturase(SEQ ID NO: 31) that was driven by the C. viswanathii ICL promoter (SEQID NO: 33). The second cassette contains codon-optimized H. armigerareductase (SEQ ID NO: 32) driven by the C. tropicalis TEF promoter (SEQID NO: 34). Gene expression in the ICL promoter cassette is terminatedby the ICL terminator sequence (SEQ ID NO: 35). Gene expression in theTEF promoter cassette is terminated by the TEF terminator sequence (SEQID NO: 36). A conservative approach was used for recoding of genes.Native gene sequences were unaltered except for replacement of CTGleucine codons with TTA. After transformation into E. coli NEB100,plasmids were miniprepped using the Zyppy Plasmid Miniprep Kit (ZymoResearch, Irvine, Calif.). Plasmids were linearized by digestion withBsiWI (New England Biolabs, Ipswich, Mass.) before transformation intoSPV053. After digestion, DNA was isolated using Clean and ConcentratorKit (Zymo Research, Irvine, Calif.). Approximately 3-5 μg of DNA wastransformed by electroporation. Positive integrants were found to besite-specific and genotyping was conducted by check PCR. A two-stageapproach was adopted for further screening of low efficiencytransformations. Approximately 100 colonies were re-patched on YPD+250μg/ml Zeocin and grown overnight. The subset of patches which grewquickly (dense growth within 24 hours) were screened by colony PCR.

Functional Expression Assay

Palmitic Acid Supplementation in YPD

Positive isolates were re-patched onto YPD+300 μg/ml Zeocin, grownovernight and then stored at 4° C. Strains were inoculated from patchplates into 2 ml of YPD in 24 deep well plates (square well, pyramidbottom). Four positive clones were inoculated for each desaturase andreductase variant and three positive clones were inoculated for eachdesaturase and mCherry expressing control strain. Deep well plates wereincubated at 30° C., 1000 rpm, and 80% humidity in the Infors HTMultitron Pro plate shaker for 24 hrs. After 24 hrs of incubation, a 1ml volume of each culture was pelleted by centrifugation at 500×g. Eachpellet was resuspended in 2 ml of YPD+0.3% (v/v) ethanol. Ethanol wasadded at this stage to induce recombinant enzyme expression from the ICLpromoter. Cultures were incubated for another 24 hours under the sameconditions before 300 mg/L palmitic acid was added to cultures from a 90g/L stock solution in ethanol. The result was the addition of a fresh0.3% ethanol in conjunction with the palmitic acid. All cultures wereincubated for an additional 24 hrs before a final addition of 0.3%ethanol. After another 24 hr period of incubation, 1.5 ml of eachculture was harvested in 1.7 ml microcentrifuge tubes and pelleted.Supernatant was removed and pellets were processed as described below.

Metabolite Extraction and GC-MS Detection

The pelleted cells (in 1.5 mL plastic tubes), usually about 10 mg to 80mg, were resuspended in methanol containing 5% (w/w) of sodiumhydroxide. The alkaline cell suspension was transferred into a 1.8 mLcrimp vial. The mixture was heated for 1 h in a heat block at 90° C.Prior to acidification with 400 μL 2.5 N HCl the vial was allowed tocool to room temperature. 500 μL chloroform containing 1 mM methylheptadecanoate were added and the mixture was shaken vigorously, thenboth aqueous and organic phase were transferred into a 1.5 mL plastictube. The mixture was centrifuged at 13,000 rpm, afterwards 450 μL ofthe organic phase were transferred into a GC vial. The organic phase wasevaporated in a heat block at 90° C. for 30 min. The residue wasdissolved in 50 μL N,O-Bis(trimethylsilyl)trifluoroacetamide containing1% trimethylchlorosilane. Prior to transfer into glass inserts themixture was heated 5 min at 90° C. The samples were analyzed by GC-MS(Table 13).

TABLE 13 Analytical parameters used for GC-MS analysis of metabolitesSystem Agilent 6890 N GC, ChemStation G1701EA E.02.01.1177 Column DB2330 m × 25 μm × 25 μm Pressure = 11.60 psi; Flow = 0.6 mL/min InletHeater = 250° C., Pressure = 11.74 psi; Total Flow {He} = 111 mL/minCarrier He @ 29 cm/sec, 11.60 psi Signal Data rate = 2 Hz/0.1 min Oven150° C. for 1 min Ramp 12° C./min to 220° C., hold 3 min Ramp 35° C./minto 300° C., hold 4 min Injection Splitless, 250° C. Detector HP 5973 MSDin SIM mode (m/z: 208.0, 297.3 and 387.3), 100 msec Dwell, EMV mode:Gain factor 1, 2.4 min solvent delay, 3.09 cycles/sec Sample Injectionvolume = 1 μL

Example 9: Insect Fatty Alcohol Production from Yarrowia lipolyticaBackground and Rationale

Yarrowia lipolytica was engineered as a production platform for insectfatty alcohol (Z11-16OH and Z9-16OH) synthesis from palmitic acid.

After individually confirming functional expression of a Z11 desaturase(Example 7) and fatty acyl-CoA reductase (FAR), the full Z11-16OH andZ9-16OH pathways (Bdr) were engineered in Y. lipolytica. For the purposeof improving fatty alcohol titers, cultivations designed for promotinggrowth vs. for eliciting lipid storage were also explored. A growthcondition favors high biomass production, but limits fatty acyl-CoA poolsize used by the engineered pathway and directs fatty acyl-CoAintermediates to membrane synthesis. Conversely, a lipid storagecondition creates a strong sink for production of fatty acyl-CoAs whichis desirable. However, fatty acyl-CoA transport towards lipid bodiescreates a strong competition for FAR activity. Under this secondscenario, even though Z11-16Acid or Z9-16Acid accumulates in the cell,most of it is inaccessible to the FAR. On the other hand, there may be acontinual flux of lipid remobilization under lipid storage conditionswhich leads to a sustained pool of Z11-16CoA or Z9-16CoA which isavailable to the FAR.

Summary of Approach

Two biodesaturation-reduction (Bch) pathway variants were tested in theH222 APAAAF (SPV300) background. The first combined recombinantexpression of Helicoverpa zea Z11 desaturase paired with a Helicoverpaarmigera fatty acyl-CoA reductase (FAR) creating a Z11-16OH synthesispathway. The second combined native Y. lipolytica Z9 desaturase activitywith H. armigera fatty acyl-CoA reductase (FAR) expression creating aZ9-16OH pathway.

Two integration plasmids were constructed to express the H. zeadesaturase and the H. armigera FAR. The TEF promoter was used fordesaturase expression and the EXP1 (export protein) or the TAL1(transaldolase) promoter was used for reductase expression.

Successful integration of the Z11-16OH pathway cassette into the H222APAAAF (SPV300) background was confirmed by colony PCR.

Functionality of the full Z11-16OH pathway was assessed via an in vivobioconversion of 16Acid (palmitic acid) into Z11-16OH(Z-11-hexadecenol).

Functionality of a full Z9-16OH pathway was assessed via an in vivobioconversion of 16Acid (palmitic acid) using previously constructedSPV471 (H222 APAAAF derived) which expresses the H. armigera FAR drivenby the TEF promoter.

GC-MS analysis was used to identify and quantify Z9-16OH and Z11-16OH.GC-FID analysis was used to identify and quantify fatty acids.

Summary

Ten isolates expressing the H. zea desaturase (pTEF) and H. armigerareductase (pEXP1) were screened. The in vivo bioconversion assayconfirmed Z11-16OH production from all isolates.

Relatively low, detectable Z11-16OH titers (0.26±0.09 mg/L) wereobserved in a YPD medium supplemented with 10 g/L methyl palmitate. TheZ11-16Acid precursor was measured at 220±11 mg/L (across clones 2, 4, 9,17, 23).

Higher Z11-16OH titers were observed in a semi-defined medium with C:Nratio of ˜80. Across all 10 isolates Z11-16OH was produced at 2.65±0.36mg/L. The Z11-16Acid precursor titer was 900±30 mg/L. One isolate(SPV578) produced 3.68±0.31 mg/L Z11-16OH (Z11-16Acid 840±14 mg/L).

Nine isolates expressing the H. zea desaturase (pTEF) and H. armigerareductase (pTAL1) were screened. The in vivo bioconversion assayconfirmed Z11-16OH production from all isolates.

One isolate (SPV603) produced 6.82±1.11 mg/L Z11-16OH in a semi-definedmedium (Z11-16Acid 1.36 g/L).

The previously tested reductase strain, SPV471 (H222 APAAAF expressingH. armigera FAR), produced 4.30±2.33 mg/L Z9-16OH and 450±80 mg/LZ9-16Acid using a semi-defined medium (C:N ratio of ˜80).

TABLE 14 Summary table of Z11/Z9-16OH titers from B_(dr) pathway strainsin in vivo bioconversion assay. Z11-16OH Z9-16OH Strain Medium (mg/L)(mg/L) pTEF-H. zea Z11 desaturase Semi-Defined 3.99 ± 0.37 0.22 ± 0.03pEXP-H. armigera FAR C:N = 80 (n = 4) (n = 4) Clone 17 (SPV578) pTEF-H.zea Z11 desaturase Semi-Defined 6.82 ± 1.11 0.22 ± 0.01 pTAL-H. armigeraFAR C:N = 80 (n = 2) (n = 2) Clone 9 (SPV603) pOLE1-Y. lipolytica OLE1Semi-Defined 0.22 ± 0.03 4.30 ± 2.23 (native) C:N = 80 (n = 2) (n = 2)pTEF-H. armigera FAR (SPV471)

Results

Strain Construction

Evidence in the literature suggests both insect desaturases and FARs arelocalized in the membrane of the endoplasmic reticulum with active sitesoriented towards the cytoplasm. Of the functional variants, the Z11desaturase from H. zea and the FAR from H. armigera were selected, onehypothesis being that using enzymes from the same genus (Helicoverpa)could better conserve protein-protein interactions that may occur in theER membrane.

Two new constructs were ordered from Genscript and cloned into thepreviously assembled H. zea desaturase plasmid, pPV0199. Two FARsynthons with either the EXP1 or TAL1 promoter from Y. lipolytica werecloned into this expression cassette.

One dual expression plasmid (with EXP1 promoter) was transformed intothe parent strain SPV300 (H222 Δpox1 Δpox2 Δpox3 Δpox4 Δpox5 Δpox6 Δadh1Δadh2 Δadh3 Δadh4 Δadh5 Δadh6 Δadh7 Δfao1 Δura3). Two differentcompetent cell preparations of the same parent strain were transformedto study variability in strain performance resulting from competent cellpreparation. Approximately 25% of URA+clones were confirmed to betargeted integrants at the XPR2 locus (20% for preparation 1, 33% forpreparation 2). Two clones from Comp. Cell Preparation 1 and all eighttargeted clones from Comp. Cell Preparation 2 were selected forscreening in the functional expression assay.

The second dual expression plasmid (with TAL1 promoter) was integratedinto the same parent strain (SPV300). Twenty-three colonies werescreened by check PCR and 11 were found to be targeted integrants (48%).Nine integrants were selected for screening in the functional expressionassay.

The construct of SPV471 (H222 ΔPΔAΔF expressing H. armigera FAR) wasdescribed previously.

Z11-16OH Functional Expression Assay

An in vivo, 24-well plate assay was used to evaluate production ofZ11-16OH. The assay was based on designs used for screening desaturaseand reductase variants as well as conditions used to increase fatty acidaccumulation. A rich medium (YPD) and a semi-defined medium were usedwith 10 g/L methyl palmitate supplemented as bioconversion substrate.The semi-defined medium had a C:N ratio of ˜80 and included 5 g/Lglycerol and 60 g/L glucose (See Materials & Methods for furtherdetails).

The initial screen of strains harboring the H. zea desaturase driven bythe TEF promoter and the H. armigera FAR driven by the EXP1 promoterconfirmed that the presence of FAR was required to produce Z11-16OH. Nohexadecenol was observed from both the parent and desaturase-onlycontrol strains under any condition. Under both media conditionsZ11-16OH and to a lesser extent Z9-16OH were detected from clonesexpressing the full desaturase-reductase pathway. When the conversionwas completed in rich medium, 0.26±0.09 mg/L Z11-16OH and 0.06±0.01 mg/LZ9-16OH were produced (FIG. 32A). A 10-fold increase in Z11-16OH titerand 3-fold increase in Z9-16OH titer was observed when the Semi-Definedmedium was used (FIG. 32B). Across all pathway clones 2.65±0.29 mg/LZ11-16OH and 0.18±0.02 mg/L Z9-16OH were produced. The enrichment ofZ11-16OH over Z9-16OH supports the potential for engineering aregiospecific Bdr pathway. Consistency between technical replicatesvaried across clones under the Semi-Defined medium condition. Titers forClones 2, 4, 6, 9, and 17 were consistent with CVs <20. Clones 1, 7, and23 have CVs >40%. The highest consistent Z11-16OH titer was observed forClone 17, 3.68±31 mg/L (Table 15).

TABLE 15 Summary table of Z11/Z9-16OH titers for pEXP1 clones. Apopulation of ten isolates expressing the H. zea desaturase driven bypTEF and H. armigera reductase driven by pEXP1, from two independentcompetent cell preparations, were assayed for Z11-16OH and Z9-16OHproduction under two different media conditions. Alcohol productionacross isolates and from select clones are presented. Z11-16OH Z9-16OHfold pTEF-Hz_desat fold increase increase pEXP-Ha_FAR Z11-16OH Z9-16OH(relative to (relative to Clone(s) Medium (mg/L) (mg/L) YPD) YPD) Allclones YPD 0.26 ± 0.09 0.06 ± 0.01 — — All clones Semi-Defined 2.65 ±0.29 0.18 ± 0.02 10 3 Clone 2 (SPV574) YPD 0.18 ± 0.09 0.05 ± 0.03 — —Clone 2 (SPV574) Semi-Defined 2.08 ± 0.26 0.14 ± 0.04 12 3 Clone 4(SPV575) YPD 0.28 ± 0.01 0.11 ± 0.01 — — Clone 4 (SPV575) Semi-Defined3.24 ± 0.28 0.21 ± 0.03 12 2 Clone 9 (SPV576) YPD 1.03 ± 0.84 0.05 ±0.01 — — Clone 9 (SPV576) Semi-Defined 1.56 ± 0.28 0.11 ± 0.02 1.5 2Clone 23 (SPV577) YPD 0.16 ± 0.14 0.05 ± 0.05 — — Clone 23 (SPV577)Semi-Defined 3.35 ± 1.85 0.26 ± 0.15 21 5 Clone 17 (SPV578) YPD 0.19 ±0.01 0.06 ± 0.01 — — Clone 17 (SPV578) Semi-Defined 3.68 ± 0.31 0.26 ±0.02 14 4

The lipid profiles of the full pathway clones were also quantified. Forsimplicity the 16 carbon fatty acid species are plotted for selectclones in FIG. 33A-33B. In general, the full Bdr pathway clonesaccumulated less Z11-16Acid than the desaturase only control (0.25<0.5g/L in YPD, 0.8-1.0<1.5 g/L in Semi-Defined). Lower Z11-16Acid titers infull Bdr pathway clones may result from reduced desaturase expression inthe dual expression cassette or potentially from Z11-16Acid consumptionby FAR and subsequent byproduct pathways. No trend in 16Acid titer wasobserved in YPD, while 16Acid titers were similar for desaturase onlyand full pathway strains in the Semi-Defined medium.

Strains using the second dual expression cassette (pTAL-Ha_FAR) wereassayed under the same Semi-Defined medium condition used to evaluatethe pEXP clones. Nine pTAL clones were assayed against SPV300 (parent),SPV575 (pEXP-Ha_FAR Clone 4), and SPV578 (pEXP-Ha_FAR Clone 17)controls. As expected, no alcohol products were observed from thenegative control. Alcohol titers from pEXP positive control strainsreplicated results observed in the initial assay of pEXP clones (FIG.34, Table 16). Excluding one outlier clone, Clone 9, Z11-16OH titer wasequivalent from pTAL clones (4.19±0.16 mg/L) and pEXP clones (4.10±0.22mg/L). Clone 9 produced Z11-16OH at 6.82±1.11 mg/L. As in the firstassay with pEXP clones, low, but detectable titers of Z9-16OH wereobserved (FIG. 34, Table 16).

TABLE 16 Summary table of Z11/Z9-16OH titers for pTAL1 clones. Apopulation of nine isolates expressing the H. zea desaturase under theTEF promoter and H. armigera reductase under the TAL promoter wereassayed for Z11-16OH and Z9-16OH production under a Semi-Defined mediumcondition. Clones were compared to positive controls expressing the H.zea desaturase under the TEF promoter and H. armigera reductase underthe EXP promoter. Alcohol production across isolates and from selectclones are presented. pTEF-Hz_desat Z11-16OH Z9-16OH pEXP-Ha_FARClone(s) Medium (mg/L) (mg/L) EXP Clone 4 (SPV575) Semi-Defined 3.91 ±0.44 0.15 ± 0.01 EXP Clone 17 (SPV578) Semi-Defined 4.30 ± 0.16 0.17 ±0.02 pTAL clones excluding Clone 9 Semi-Defined 4.19 ± 0.16 0.18 ± 0.01pTAL Clone 9 (SPV603) Semi-Defined 6.82 ± 1.11 0.22 ± 0.01

The lipid profiles of all strains in the second (pTAL) full pathwayscreen were also quantified. For simplicity the 16 carbon fatty acidspecies are plotted in FIG. 35. As expected, Z11-16Acid is present onlyfor strains expressing the desaturase. Complete lipid profiles weresimilar to those observed previously (FIG. 36). Z9-18Acid (oleic acid)was the second most abundant fatty acid species after Z11-16Acid.

Z9-16OH Functional Expression Assay

An in vivo, flask scale assay was used to test for Z9-16OH production.The parent control strain, H222 APAAAF (SPV300), was compared to astrain expressing H. armigera FAR which relied on native Z9 desaturaseactivity to synthesize the Z9-16CoA precursor (SPV471). Biomass wasgenerated through a YPD seed culture, mimicking the plate assay.Bioconversion flasks were inoculated at an initial OD600=1 or OD600=4into the same Semi-Defined C:N=80 medium used in the Z11-16OH plateassay (See Materials & Methods for details). As expected, control flasksdid not produce detectable Z9-16OH while SPV471 flasks produced up to4.30±2.23 mg/L after 24 hours of incubation (FIG. 37A-FIG. 37B). Whilethere was large variability between replicates, all SPV471 (H. armigeraFAR) replicates exceeded 1 mg/L titer. Increased seeding density did notincrease Z9-16Acid or Z9-16OH titer. The precursor Z9-16Acid titer at 24hours was significantly less (<0.5 g/L) than the Z11-16Acid precursorobserved for dual expression cassette strains used to produce Z11-16OH.The relative abundance of other fatty acid species was similar topreviously observed profiles, with Z9-18Acid as the next most abundantspecies (FIG. 38). Both lipid and alcohol samples were taken over thecourse of 48 hours to produce a time course of Z9-16OH and lipid titers.Z9-16OH titer peaked at 24 hours before decreasing over the second day(FIG. 39A). Z9-16Acid increased rapidly over the first 24 hours beforestabilizing or increasing slowly over the second 24 hours (FIG. 39B).Since the employed analytical method utilizes only the cell pellet, thedecrease in Z9-16OH titer supports the hypothesis of downstreamconsumption or secretion of the alcohol products. They may be oxidized(w-oxidation), secreted as free alcohol, or derivatized and secreted asan ester. Analysis of supernatant samples using FID and MS SCANdetection revealed no detectable Z9-16OH or Z9-16OH derivativessupporting the hypothesis of consumption via oxidation pathways.

Conclusions

Combining expression of Helicoverpa Z11 desaturase and fatty acyl-CoAreductase led to production of Z11-16OH in Y. lipolytica H222 APAAAF(SPV300) at titers >1 mg/L.

High C:N ratio conditions improved Z11-16OH titer relative to a richmedium condition.

Under lipid accumulating conditions the combination of native Z9desaturase and H. armigera FAR activities are sufficient for synthesisof >1 mg/L Z9-16OH.

Titers are increased, for example, by deleting pathways consuming fattyalcohol products and/or fatty acid precursors; identifying FAR variantswhich exhibit higher turn-over rate than H. armigera FAR; and/orincreasing pathway copy number.

Key undesired byproducts are identified.

The possibility that some of the fatty alcohol product is converted intofatty acetate by the activity of one or more endogenousacetyltransferases is explored.

Improved host strains are engineered to eliminate the w-oxidationpathway and components of the lipid storage pathway.

Additional copies of desaturase and FAR are integrated into Y.lipolytica.

Materials & Methods

Strain Construction

All desaturase and reductase genes were ordered from Genscript. Homosapiens codon optimization was used (Genscript algorithm). The newlysynthesized expression cassette was subcloned into pPV199 by Genscriptusing the SapI restriction site. Plasmids were transformed and preppedfrom E. coli EPI400 using the Zyppy Plamsid Miniprep Kit (Zymo Research,Irvine, Calif.). Plasmids were digested with PmeI (New England Biolabs,Ipswich, Mass.) and purified by gel extraction using Zymoclean Gel DNArecovery Kit (Zymo Research, Irvine, Calif.). DNA was furtherconcentrated using Clean and Concentrator Kit (Zymo Research, Irvine,Calif.). Approximately ˜1-2 μg of DNA was transformed using Frozen-EZYeast Transformation II Kit (Zymo Research, Irvine, Calif.). Themanufacturer's protocol was modified as follows: A 2 ml YPD seed culturewas inoculated at 9 am the day before competent cell preparation. Theseed was grown 8 hours (until 5 pm) before 40 ml of YPD in a 250 mlbaffled shake flask (or 20 ml in a 125 ml baffled flask) was inoculatedto an initial OD600 of 0.0005. The culture was incubated at 28° C. and250 rpm ˜24 hours. Cells were harvested at an OD600=0.5-1. Instead ofresuspending 10 ml of culture in 1 ml of Solution 2 as in themanufacturer's instructions (OD600˜10), 10 ml of SPV140 culture wasresuspended in 0.5 ml (OD600˜20-30). All Solution 2 aliquots were slowlyfrozen to −80° C. by placing the tubes in a closed Styrofoam box beforeputting in the −80° C. freezer. 50 μl aliquots of competent cells in 1.7ml Eppendorf tubes were thawed on ice, DNA eluted in water was addeddirectly to the cells, and 500 μl of Solution 3 was used to suspend thecells with gentle pipetting. Tubes were incubated at 28° C. for 3 hourswith gentle vortexing every 30 minutes. The entire transformationmixture was plated on CM glucose -ura agar plates. Positive integrantswere found to be site-specific and genotyping was conducted by checkPCR.

Z11-16OH Functional Expression Assay

Positive isolates were repatched onto YPD, grown overnight, and thenstored at 4° C. Strains were inoculated from patch plates into 2 ml ofYPD in 24 deepwell plates (square well, pyramid bottom). Replicateinoculations were made from each patch. Negative control strains werestruck out on YPD from glycerol stocks and individual colonies were usedto inoculate. Deepwell plates were incubated at 28° C. and 250 rpm inthe Infors Multitron refrigerated flask shaker for 24 hrs. After 24 hrsof incubation, a 0.85 ml volume of each culture was pelleted bycentrifugation at 800×g. Each pellet was resuspended in either 2 ml ofYPD or Semi-defined medium (described in Table 17 below). 10 g/L methylpalmitate (pre-warmed to ˜50° C.) was added to cultures. All cultureswere incubated for 48 hours before endpoint sampling. Final celldensities were measured with the Tecan Infinite 200pro plate reader. 1.5ml (alcohol analysis) or 500 μl (lipid analysis) was transferred to 1.7ml microcentrifuge tubes and pelleted. Supernatant was transferred toclean tubes and samples were processed as described below.

TABLE 17 Semi-defined (C:N = 80) medium composition. Components of thesemi-defined base medium used to induce lipid storage are described.Media Components Conc. Units Yeast Extract 2 g/L Peptone 1 g/L Potassiumphosphate buffer pH7 0.1 M YNB w/o aa, NH4 1.7 g/L Glucose 60 g/LGlycerol 5 g/l

Z9-16OH Functional Expression Assay

SPV300 (negative control) and SPV471 were struck out onto YPD agarplates, grown overnight, and then stored at 4° C. Strains wereinoculated from colonies into 2 ml of YPD and incubated at 28° C. and250 rpm in 14 ml round bottom culture tubes for ˜8 hours. Afterincubation, 2 ml of culture was used to inoculate 20 ml of YPD in a 125ml baffled shake flask. Shake flasks were incubated 24 hrs at 28° C. and250 rpm. After incubation, cell density in shake flasks was measuredusing a Tecan Infinite 200pro plate reader. An appropriate volume ofculture was pelleted in order to resuspend cells in 25 ml ofSemi-defined C:N=80 medium (see Table 17 above) at an initial OD600=1(˜1 gDCW/L) or 4 (˜4 gDCW/L). The resuspended culture was added to 250ml baffled shake flasks. Neat methyl palmitate was added at 10 g/L finalconcentration after pre-heating to 50° C. After substrate addition,flasks were incubated at 28° C. and 250 rpm for two days. At 12, 18, 24,36, 42, and 48 hours 500 μl (lipid analysis) and 1.5 ml (alcoholanalysis) samples were taken in 1.7 ml microcentrifuge tubes. Sampleswere pelleted and the supernatant was transferred to a cleanmicrocentrifuge tube.

Metabolite Extraction and GC-MS Detection

Alcohol Analysis

The pelleted cells (in 1.5 mL plastic tubes), usually about 10 mg to 80mg, were resuspended in methanol containing 5% (w/w) of sodiumhydroxide. The alkaline cell suspension was transferred into a 1.8 mLcrimp vial. The mixture was heated for 1 h in the heat block at 90° C.Prior to acidification with 400 μL 2.5 N HCl the vial was allowed tocool to room temperature. 500 μL chloroform containing 1 mM methylheptadecanoate were added and the mixture was shaken vigorously, thenboth aqueous and organic phase were transferred into a 1.5 mL plastictube. The mixture was centrifuged at 13,000 rpm, afterwards 450 μL ofthe organic phase were transferred into a GC vial. The organic phase wasevaporated in a heat block at 90° C. for 30 min. The residue wasdissolved in 50 μL N,O-Bis(trimethylsilyl)trifluoroacetamide containing1% trimethylchlorosilane. Prior to transfer into glass inserts themixture was heated 5 min at 90° C. The samples were analyzed by GC-MS(Table 18).

TABLE 18 GC-MS parameters System Agent 6890 N GC, ChemStation G1701EAE.02.01.1177 Column DB23 30 m × 25 μm × 25 μm Pressure = 11.50 psi; Flow= 0.6 mL/min Inlet Heater = 250° C.; Pressure = 11.74 psi; Total Flow{He} = 111 mL/min Carrier He @ 29 cm/sec, 11.60 psi Signal Data rate = 2Hz/0.1 min Oven 150° C. for 1 min Ramp 12° C./min to 220° C., hold 3 minRamp 35° C./min to 300° C., hold 4 min Injection Splitless, 250° C.Detector Initial strain screening and first technical triplicate: HP5973 MSD in SIM mode (m/z: 208.0, 297.3 and 387.3), SPV488/SPV490alcohol quantification: HP 5973 MSD in SIM mode (m/z: 284.0 and 297.3),100 msec Dwell, EMV mode: Gain factor 1, 2.4 min solvent delay, 3.09cycles/sec Sample Injection volume = 1 uL

Lipid Analysis

Total lipid composition was based on modified procedures by Moss et al.(1982) and Yousuf et al (2010). The pelleted cells (in 1.5 mL plastictubes), usually about 10 mg to 80 mg, were resuspended in methanolcontaining 5% (w/w) of sodium hydroxide. The alkaline cell suspensionwas transferred into a 1.8 mL glass crimp GC-vial. The mixture washeated for 1 h in the heat block at 90° C. Prior to acidification with400 μL 2.5 N HCl, the vial was allowed to cool to room temperature. 500μL chloroform containing 1 mM methyl heptadecanoate were added and themixture was shaken vigorously, then both aqueous and organic phase weretransferred into a 1.5 mL plastic tube. The mixture was centrifuged at13,000 rpm, afterwards 450 μL of the organic phase was transferred intoa new 1.8 mL glass screw-cap GC-vial. After cooling to room temperatureresidual fatty acid methyl esters and free fatty acids were dissolvedand derivatized in methanol containing 0.2 M TMSH (trimethylsulfoniumhydroxide)(Table 19).

TABLE 19 GC-MS parameters System Agilent 6890 GC, ChemStation Rev.B.03.02 (341) Column J&W DB-23 30 m × 25 mm × 25 μm Pressure = 16 psi;Flow = 0.9 mL/min; Run Time = 14.4 min Inlet Heater = 240° C.; Pressure=16 psi; Total Flow {He} = 31.4 mL/min Carrier H₂ @ 1 mL/min, 9 psi, 35cm/sec Signal Data rate = 2 Hz/0.1 min Oven 150° C. for 1 min Ramp 12°C./min to 220° C., hold 3 min Ramp 35° C./min to 240° C., hold 6 minEquilibration Time: 2 min Injection Split, 240° C. Split ratio - 30:1;29.1 mL/min Detector FID, 240° C. H₂ @ 35.0 mL/min, Air @ 350 mL/min;;Electrometer {Lit Offset} @ 2.0 pA Sample Injection volume = 1 uL

SEQUENCE LISTING SEQ ID NO: 1Agrotis segetum FAR_S. cerevisiae codon optATGCCAGTTTTGACTTCTAGAGAAGATGAAAAGTTGTCAGTTCCAGAATTTTACGCTGGTAAATCTATCTTCGTTACAGGTGGTACTGGTTTCTTGGGTAAAGTTTTTATTGAAAAGTTGTTGTACTGTTGTCCAGATATTGATAAAATCTATATGTTAATTAGAGAAAAGAAAAATTTGTCTATTGATGAAAGAATGTCAAAGTTOTTGGATGATCCATTATTTTCTAGATTGAAGGAAGAAAGACCTGGTGACTTGGAAAAGATTGTTTTGATTCCAGGTGACATTACAGCTCCAAATTTGGGTTTATCAGCAGAAAACGAAAGAATTTTGTTAGAAAAAGTTTCTGTTATTATTAATTCAGCTGCAACTGTTAAGTTTAATGAACCATTGCCAATCGCTTGGAAGATTAATGTTGAAGGTACAAGAATGTTGTTGGCATTGTCTAGAAGAATGAAGAGAATCGAAGTTTTTATTCATATTTCTACTGCTTACTCAAATGCATCTTCAGATAGAATCGTTGTTGATGAAATCTTGTATCCAGCTCCAGCAGATATGGATCAAGTTTACCAATTGGTTAAAGATGGTGTTACAGAAGAAGAAACTGAAAGATTGTTGAACGGTTTGCCAAACACTTACACTTTTACTAAGGCTTTGACAGAACATTTGGTTGCAGAACATCAAACATACGTTCCAACTATCATCATCAGACCATCTGTTGTTGCTTCAATTAAAGATGAACCAATCAGAGGTTGGTTATGTAATTGGTTTGGTGCTACAGGTATCTCTGTTTTTACTGCAAAGGGTTTGAACAGAGTTTTGTTGGGTAAAGCTTCAAACATCGTTGATGTTATCCCAGTTGATTACGTTGCAAATTTGGTTATTGTTGCTGGTGCAAAATCTGGTGGTCAAAAATCAGATGAATTAAAGATCTATAACTGTTGTTCTTCAGATTGTAACCCAGTTACTTTGAAGAAAATTATTAAAGAGTTTACTGAAGATACTATTAAAAATAAGTCTCATATTATGCCATTGCCAGGTTGGTTCGTTTTTACTAAGTACAAGTGGTTGTTGACATTGTTAACTATTATTTTTCAAATGTTACCAATGTATTTGGCTGATGTTTACAGAGTTTTGACAGGTAAAATCCCAAGATACATGAAGTTGCATCATTTGGTTATTCAAACAAGATTGGGTATCGATTTCTTTACTTCTCATTCATGGGTTATGAAGACAGATAGAGTTAGAGAATTATTCGGTTCTTTGTCATTGGAGAAAAAGCATATGTTTCCATGTGATCCATCTTCAATCGATTGGACAGATTATTTGCAATCATACTGTTACGGTGTTAGAAGATTTTTGGAAAAGAAGAAATAA SEQ ID NO: 2Spodoptera littoralis FAR1_S. cerevisiae codon optATGGTTGTTTTGACTTCAAAGGAAAAATCAAACATGTCTGTTGCTGATTTCTACGCTGGTAAATCTGTTTTTATTACAGGTGGTACTGGTTTCTTGGGTAAAGTTTTTATTGAAAAGTTGTTGTACTCATGTCCAGATATTGATAAAATCTATATGTTGATCAGAGAAAAGAAAGGTCAATCTATCAGAGAAAGATTAACTAAAATTGTTGATGATCCATTGTTTAATAGATTGAAGGATAAGAGACCAGATGATTTGGGTAAAATCGTTTTGATCCCAGGTGACATCACAGTTCCAGGTTTGGGTATTTCTGAAGAAAACGAAACAATCTTGACTGAAAAAGTTTCAGTTGTTATTCATTCTGCTGCAACTGTTAAGTTTAATGAACCATTGGCTACTGCATGGAACGTTAACGTTGAAGGTACAAGAATGATCATGGCATTATCAAGAAGAATGAAGAGAATCGAAGTTTTTATTCATATTTCTACTGCTTACACTAACACAAACAGAGCAGTTATTGATGAAGTTTTGTATCCACCACCAGCTGATATCAACGATGTTCATCAACATGTTAAAAATGGTGTTACAGAAGAAGAAACTGAAAAGATTTTGAACGGTAGACCAAACACTTACACTTTTACTAAGGCTTTGACTGAACATTTGGTTGCAGAAAACCAATCATACATGCCAACAATCATTGTTAGACCATCTATTGTTGGTGCTATTAAAGATGATCCAATTAGAGGTTGGTTGGCTAATTGGTATGGTGCAACAGGTTTGTCAGTTTTTACTGCAAAGGGTTTGAACAGAGTTATATATGGTCATTCTAACCATGTTGTTGATTTGATTCCAGTTGATTACGTTGCTAATTTGGTTATTGTTGCTGGTGCALAGACATACCATTCAAACGAAGTTACTATCTATAACTCTTGTTCTTCATCTTGTAACCCAATCACTATGAAGAGATTGGTTGGTTTGTTTATTGATTACACAGTTAAGCATAAGTCATACGTTATGCCATTGCCAGGTTGGTATGTTTACTCTAACTACAAGTGGTTGGTTTTCTTGGTTACTGTTATTTTCCAAGTTATTCCAGCTTACTTAGGTGACATTGGTAGAAGATTGTTAGGTAAAAATCCAAGATACTACAAGTTGCAAAATTTGGTTGCTCAAACACAAGAAGCAGTTCATTTCTTTACATCACATACTTGGGAAATTAAATCAAAGAGAACTTCTGAATTGTTTTCATCTTTGTCTTTGACAGATCAAAGAATGTTTCCATGTGATGCTAACAGAATCGATTGGACAGATTACATCACTGATTACTGTTCTGGTGTTA GACAATTTTTGGAAAAGATTAAATAASEQ ID NO: 3 Helicoverpa armigera FAR3_S. cerevisiae codon optATGGTTGTTTTGACTTCAAAGAAACAAAGCCATCTGTTGCTGAATTTTACGCTGGTAAATCAGTTTTTATTACAGGTGGTACTGGTTTCTTGGGTAAAGTTTTTATTGAAAAGTTGTTGTACTCTTGTCCAGATATTGAAAATATCTATATGTTGATCAGAGAAAAGAAAGGTTTGTCAGTTTCTGAAAGAATTAAACAATTTTTAGATGATCCATTGTTTACAAGATTGAAGGATAAGAGACCAGCTGATTTGGAAAAGATTGTTTTGATCCCAGGTGACATCACTGCACCAGATTTGGGTATTAATTCTGAAAACGAAAAGATGTTGATTGAAAAAGTTTCAGTTATTATTCATTCTGCTGCAACTGTTAAGTTTAATGAACCATTACCAACAGCTTGAAAGATTAATGTTGAAGGTACTAGAATGATGTTGGCATTGTCAAGAAGAATGAAGAGAATCGAAGTTTTTATTCATATTTCTACAGCTTACACTAACACAAACAGAGAAGTTGTTGATGALATCTTGTATCCAGCTCCAGCAGATATCGATCAAGTTCATCAATACGTTAAGGATGGTATCTCAGAAGAAGATACTGAAAAGATTTTGAACGGTAGACCAAACACTTACACTTTTACTAAGGCTTTGACAGAACATTTGGTTGCTGAAAATCAAGCATACGTTCCAACTATTATTGTTAGACCATCTGTTGTTGCTGCAATTAAAGATGAACCATTGAAAGGTTGGTTGGGTAATTGGTTTGGTGCTACAGGTTTGACTGTTTTTACAGCAAAGGGTTTGAACAGAGTTATATATGGTCATTCTTCATACATCGTTGATTTGATCCCAGTTGATTACGTTGCTAATTTGGTTATTGCTGCAGGTGCAAAATCTTCAAAGTCAACAGAATTGAAGGTTTACAACTGTTGTTCTTCATCTTGTAACCCAGTTACTATCGGTACATTGATGTCAATGTTCGCTGATGATGCAATTAAACAAAAATCTTACGCTATGCCATTGCCAGGTTGGTACATTTTTACAAAGTACAAGTGGTTGGTTTTGTTGTTGACATTTTTGTTCCAAGTTATTCCAGCATACGTTACTGATTTGTCAAGACATTTGATCGGTAAATCTCCAAGATACATCAAGTTGCAATCATTGGTTAACCAAACTAGATCATCTATCGATTTCTTTACAAACCATTCTTGGGTTATAAAAGCTGATAGAGTTAGAGAATTGTACGCTTCATTGTCTCCAGCTGATAAGTACTTATTCCCATGTGATCCAACTGATATCAACTGGACACATTACATCCAAGATTACTGTTGGGGTGTTAGACATTTCTTGGAAAAGALATCTTACGAATAA SEQ ID NO: 4 pOLE1 cassetteCTTGCTGAAAAGATGATGTTCTGAGGTATTCGTATCGCTAGCTTGATACGCTTTTAACAAAAGTAAGCTTTTCGTTTGCAGGTTTGGTTACTTTTCTGTACGAGATGATATCGCTAAGTTTATAGTCATCTGTGAAATTTCTCAAAAACCTCATGGTTTCTCCATCACCCATTTTTCATTTCATTTGCCGGGCGAGGAAAAAAAAAAAGGAAAAAAAAAAAAAAAAATAAATGACACATGGAAATAAGTCAAGGATTAGCGGATATGTAGTTCCAGTCCGGGTTATACCATCACGTGATAATAAATCCAAATGAGAATGAGGGTGTCATATCTAATCATTATGCACGTCAAGATTCTCCGTGACTATGGCTCTTTTCTGAAGCATTTTTCGGGCGCCCGGTGGCCAAAAACTAACTCCGAGCCCGGGCATGTCCCGGGGTTAGCGGGCCCAACAAAGGCGCTTATCTGGTGGGCTTCCGTAGAAGAAAAAAAGCTGTTGAGCGAGCTATTTCGGGTATCCCAGCCTTCTCTGCAGACCGCCCCAGTTGGCTTGGCTCTGGTGCTGTTCGTTAGCATCACATCGCCTGTGACAGGCAGAGGTAATAACGGCTTAAGGTTCTCTTCGCATAGTCGGCAGCTTTCTTTCGAACGTTGAACACTCAACAAACCTTATCTAGTGCCCAACCAGGTGTGCTTCTACGAGTCTTGCTCACTCAGACACACCTATCCCTATTGTTACGGCTATGGGGATGGCACACAAAGGTGGAAATAATAGTAGTTAACAATATATGCAGAAAATCATCGGCTCCTGGCTCATCGAGTCTTGCAAATCAGCATATACATATATATATGGGGGCAGATCTTGATTCATTTATTGTTCTATTTCCATCTTTCCTACTTCTGTTTCCGTTTATATTTTGTATTACGTAGAATAGAACATCATAGTAATAGATAGTTGTGGTGATCATATTATAAACAGCACTAAAACATTACAACAAAGAATGCCAACTTCTGGAACTACTATTGAATTGATTGACGACCAATTTCCAAAGGATGACTCTGCCAGCAGTGGCATTGTCGACACTAGTGCGGCCGCTCACATATGAAAGTATATACCCGCTTTTGTACACTATGTAGCTATAATTCAATCGTATTATTGTAGCTCCGCACGACCATGCCTTAGAAATATCCGCAGCGCG SEQ ID NO: 5Extended OLE1 promoter regionCTTGCTGAAAAGATGATGTTCTGAGGTATTCGTATCGCTAGCTTGATACGCTTTTAACAAAAGTAAGCTTTTCGTTTGCAGGTTTGGTTACTTTTCTGTACGAGATGATATCGCTAAGTTTATAGTCATCTGTGAAATTTCTCAAAAACCTCATGGTTTCTCCATCACCCATTTTTCATTTCATTTGCCGGGCGGAAAAAAAAAAAAAGGAAAAAAAAAAAAAAAAATAAATGACACATGGAAATAAGTCAAGGATTAGCGGATATGTAGTTCCAGTCCGGGTTATACCATCACGTGATAATAAATCCAAATGAGAATGAGGGTGTCATATCTAATCATTATGCACGTCAAGATTCTCCGTGACTATGGCTCTTTTCTGAAGCATTTTTCGGGCGCCCGGTGGCCAAAAACTAACTCCGAGCCCGGGCATGTCCCGGGGTTAGCGGGCCCAACAAAGGCGCTTATCTGGTGGGCTTCCGTAGAAGAAAAAAAGCTGTTGAGCGAGCTATTTCGGGTATCCCAGCCTTCTCTGCAGACCGCCCCAGTTGGCTTGGCTCTGGTGCTGTTCGTTAGCATCACATCGCCTGTGACAGGCAGAGGTAATAACGGCTTAAGGTTCTCTTCGCATAGTCGGCAGCTTTCTTTCGGACGTTGA SEQ ID NO: 6OLE1 promoter regionACACTCAACAAACCTTATCTAGTGCCCAACCAGGTGTGCTTCTACGAGTCTTGCTCACTCAGACACACCTATCCCTATTGTTACGGCTATGGGGATGGCACACAAAGGTGGAAATAATAGTAGTTAACAATATATGCAGCAAATCATCGGCTCCTGGCTCATCGAGTCTTGCAAATCAGCATATACATATATATATGGGGGCAGATCTTGATTCATTTATTGTTCTATTTCCATCTTTCCTACTTCTGTTTCCGTTTATATTTTGTATTACGTAGAATAGAACATCATAGTAATAGATAGTTGTGGTGATCATATTATAAACAGCACTAA AACATTACAACAAAGASEQ ID NO: 7 OLE1 27aa leaderATGCCAACTTCTGGAACTACTATTGAATTGATTGACGACCAATTTCCAAAGGATGACTCTGCCAGCAGTGGCATTGTCGAC SEQ ID NO: 8 Vsp13 terminator regionTCACATATGAAAGTATATACCCGCTTTTGTACACTATGTAGCTATAATTCAATCGTATTATTGTAGCTCCGCACGACCATGCCTTAGAAATATCCGCAGCGCG SEQ ID NO: 9T. ni desaturase ATGGCTGTGATGGCTCAAACAGTACAAGAAACGGCTACAGTGTTGGAAGAGGAAGCTCGCACAGTGACTCTTGTGGCTCCAAAGACAACGCCAAGGAAATATAAATATATATACACCAACTTTCTTACATTTTCATATGCGCATTTAGCTGCATTATACGGACTTTATTTGTGCTTCACCTCTGCGAAATGGGAAACATTGCTATTCTCTTTCGTACTCTTCCACATGTCAAATATAGGCATCACCGCAGGGGCTCACCGACTCTGGACTCACAAGACTTTCAAAGCCAAATTGCCTTTGGAAATTGTCCTCATGATATTCAACTCTTTAGCCTTTCAAAACACGGCTATTACATGGGCTAGAGAACATCGGCTACATCACAAATACAGCGATACTGATGCTGATCCCCACAATGCGTCAAGAGGGTTCTTCTACTCGCATGTTGGCTGGCTATTAGTAAAAAAACATCCCGATGTCCTGAAATATGGAAAAACTATAGACATGTCGGATGTATACAATAATCCTGTGTTAAAATTTCAGAAAAAGTACGCAGTACCCTTAATTGGAACAGTTTGTTTTGCTCTGCCAACTTTGATTCCAGTCTACTGTTGGGGCGAATCGTGGAACAACGCTTGGCACATAGCCTTATTTCGATACATATTCAATCTTAACGTGACTTTCCTAGTCAACAGTGCTGCGCATATCTGGGGGAATAAGCCTTATGATAAAAGCATCTTGCCCGCTCAAAACCTGCTGGTTTCCTTCCTAGCAAGTGGAGAAGGCTTCCATAATTACCATCACGTCTTTCCATGGGATTACCGCACAGCAGAATTAGGGAATAACTTCCTGAATTTGACGACGCTGTTCATTGATTTTTGTGCCTGGTTTGGATGGGCTTATGACTTGAAGTCTGTATCAGAGGATATTATAAAACAGAGAGCTAAACGAACAGGTGACGGTTCTTCAGGGGTCATTTGGGGATGGGACGACAAAGACATGGACCGCGATATAAAATCTAAAGCTAACATTTTTTATGCTAAAAAGG AATGASEQ ID NO: 10 A. segetum desaturaseATGGCTCAAGGTGTCCAAACAACTACGATATTGAGGGAGGAGGAGCCGTCATTGACTTTCGTGGTACCTCAAGAACCGAGAAAGTATCAAATCGTGTACCCAAACCTTATCACATTTGGGTACTGGCATATAGCTGGTTTATACGGGCTATATTTGTGCTTTACTTCGGCAAAATGGCAAACAATTTTATTCAGTTTCATGCTCGTTGTGTTAGCAGAGTTGGGAATAACAGCCGGCGCTCACAGGTTATGGGCCCACAAAACATATAAAGCGAAGCTTCCCTTACAAATTATCCTGATGATACTGAACTCCATTGCCTTCCAAAATTCCGCCATTGATTGGGTGAGGGACCACCGTCTCCATCATAAGTACAGTGACACTGATGCAGACCCTCACAATGCTACTCGTGGTTTCTTCTATTCTCATGTTGGATGGTTGCTCGTAAGAAAACATCCAGAAGTCAAGAGACGTGGAAAGGAACTTGACATGTCTGATATTTACAACAATCCAGTGCTGAGATTTCAAAAGAAGTATGCTATACCCTTCATCGGGGCAATGTGCTTCGGATTACCAACTTTTATCCCTGTTTACTTCTGGGGAGAAACCTGGAGTAATGCTTGGCATATCACCATGCTTCGGTACATCCTCAACCTAAACATTACTTTCCTGGTCAACAGTGCTGCTCATATCTGGGGATACAAACCTTATGACATCAAAATATTGCCTGCCCAAAATATAGCAGTTTCCATAGTAACCGGCGGCGAAGTTTCCATAACTACCACCACGTTTTTTCCTTGGGATTATCGTGCAGCAGAATTGGGGAACAATTATCTTAATTTGACGACTAAGTTCATAGATTTCTTCGCTTGGATCGGATGGGCTTACGATCTTAAGACGGTGTCCAGTGATGTTATAAAAAGTAAGGCGGAAAGAACTGGTGATGGGACGAATCTTTGGGGTTTAGAAGACAAAGGTGAAG AAGATTTTTTGAAAATCTGGAAAGACAATTAA SEQ ID NO: 11T. pseudonana desaturase ACTAGTATGGACTTTCTCTCCGGCGATCCTTTCCGGACACTCGTCCTTGCAGCACTTGTTGTCATCGGATTTGCTGCGGCGTGGCAATGCTTCTACCCGCCGAGCATCGTCGGCAAGCCTCGTACATTAAGCAATGGTAAACTCAATACCAGAATCCATGGCAAATTGTACGACCTCTCATCGTTTCAGCATCCAGGAGGCCCCGTGGCTCTTTCTCTTGTTCAAGGTCGCGACGGAACAGCTCTATTTGAGTCACACCATCCCTTCATACCTCGAAAGAATCTACTTCAGATCCTCTCCAAGTACGAGGTTCCGTCGACTGAAGACTCTGTTTCCTTCATCGCCACCCTAGACGAACTCAATGGTGAATCTCCGTACGATTGGAAGGACATTGAAAATGATGATTTCGTATCTGACCTACGAGCTCTCGTAATTGAGCACTTTTCTCCTCTCGCCAAGGAAAGGGGAGTTTCACTCGTTGAGTCGTCGAAGGCAACACCTCAGCGGTGGATGGTGGTTCTACTGCTCCTTGCGTCGTTCTTCCTCAGCATCCCATTATATTTGAGTGGTTCGTGGACTTTCGTTGTCGTCACTCCCATCCTCGCTTGGCTGGCGGTTGTCAATTACTGGCACGATGCTACTCACTTTGCATTGAGCAGCAACTGGATTTTGAATGCTGCGCTCCCATATCTCCTCCCTCTCCTATCGAGTCCGTCAATGTGGTATCATCATCACGTCATTGGACATCACGCATACACCAACATTTCCAAAAGAGATCCAGATCTTGCTCACGCTCCACAACTCATGAGAGAACACAAGAGTATCAAATGGAGACCATCTCACTTAAATCAAACACAGCTTCCGCGGATTCTCTTCATCTGGTCGATTGCAGTCGGTATTGGGTTGAACTTACTGAACGACGTGAGAGCACTAACCAAGCTTTCATACAACAACGTTGTTCGGGTGGAGAAGATGTCATCGTCGCGAACATTACTCCATTTCCTTGGACGTATGTTGCACATCTTTGTGACTACACTTTGGCCCTTTTTGGCGTTTCCGGTGTGGAAGGCCATCGTTTGGGCGACTGTACCGAATGCCATACTGAGTTTGTGCTTCATGCTGAATACGCAAATCAATCACCTCATCAACACGTGTGCACATGCTTCCGATAACAACTTTTACAAGCATCAAGTTGTAACTGCTCAGAACTTTGGCCGATCAAGTGCCTTTTGCTTCATCTTCTCGGGAGGTCTCAACTACCAAATTGAACATCATTTGTTGCCGACGGTGAACCATTGCCATTTGCCAGCTTTGGCCCCGGGTGTAGAGCGTTTGTGTAAGAAACACGGGGTGACATACAACTCTGTTGAAGGATACAGAGAGGCCATCATTGCACACTTTGCACATACCAAAGATATGTCGACGAAGCCTACTG ATTGA SEQ ID NO: 12A. transitella desaturaseATGGTCCCTAACAAGGGTTCCAGTGACGTTTTGTCTGAACATTCTGAGCCCCAGTTCACTAAACTCATAGCTCCACAAGCAGGGCCGAGGAAATACAAGATAGTGTATCGAAATTTGCTCACATTCGGCTATTGGCACTTATCAGCTGTTTATGGGCTCTACTTGTGCTTTACTTGTGCGAAATGGGCTACCATCTTATTTGCATTTTTCTTATACGTGATCGCGGAAATCGGTATAACAGGTGGCGCTCATAGGCTATGGGCACATCGGACTTATAAAGCCAAGTTGCCTTTAGAGATTTTGTTACTCATAATGAACTCTATTGCCTTCCAAGACACTGCTTTCACCTGGGCTCGTGATCACCGCCTTCATCACAAATATTCGGATACTGACGCTGATCCCCACAATGCTACCAGAGGGTTTTTCTATTCACATGTAGGCTGGCTTTTGGTGAAGAAACACCCTGAAGTCAAAGCAAGAGGAAAATACTTGTCGTTAGATGATCTTAAGAATAATCCATTGCTTAAATTCCAAAAGAAATACGCTATTCTAGTTATAGGCACGTTATGCTTCCTTATGCCAACATTTGTGCCCGTATACTTCTGGGGCGAGGGCATCAGCACGGCCTGGAACATCAATCTATTGCGATACGTCATGAATCTTAACATGACTTTCTTAGTTAACAGTGCAGCGCATATCTTTGGCAACAAACCATACGATAAGAGCATAGCCTCAGTCCAAAATATTTCAGTTAGCTTAGCTACTTTTGGCGAAGGATTCCATAATTACCATCACACTTACCCCTGGGATTATCGTGCGGCAGAATTAGGAAATAATAGGCTAAATATGACTACTGCTTTCATAGATTTCTTCGCTTGGATCGGCTGGGCTTATGACTTGAAGTCTGTGCCACAAGAGGCCATTGCAAAAAGGTGTGCGAAAACTGGCGATGGAACGGATATGTGGGGTCGAAAA AGATAASEQ ID NO: 13 H. zea desaturaseATGGCCCAAAGCTATCAATCAACTACGGTTTTGAGTGAGGAGAAAGAACTAACACTGCAACATTTGGTGCCCCAAGCATCGCCCAGGAAGTATCAAATAGTGTATCCGAACCTCATTACGTTTGGTTACTGGCACATAGCCGGACTTTATGGCCTTTACTTGTGCTTCACTTCTGCTAAATGGGCTACGATTTTATTCAGCTACATCCTCTTCGTGTTAGCAGAAATAGGAATCACGGCTGGCGCTCACAGACTCTGGGCCCACAAAACTTACAAAGCGAAACTACCATTAGAAATACTCTTAATGGTATTCAACTCCATCGCTTTTCAAAACTCAGCCATTGACTGGGTGAGGGACCACCGACTCCACCATAAGTATAGCGATACAGATGCTGATCCCCACAATGCCAGCCGAGGGTTCTTTTATTCCCATGTAGGATGGCTACTTGTGAGAAAACATCCTGAAGTCAAAAAGCGAGGGAAAGAACTCAATATGTCCGATATTTACAACAATCCTGTCCTGCGGTTTCAGAAAAAATACGCCATACCCTTCATTGGGGCTGTTTGTTTCGCCTTACCTACAATGATACCTGTTTACTTCTGGGGAGAAACCTGGTCCAATGCTTGGCATATCACCATGCTTCGCTACATCATGAACCTCAATGTCACCTTTTTGGTAAACAGCGCTGCTCATATATGGGGAAACAAGCCTTATGACGCAAAAATATTACCTGCACAAAATGTAGCTGTGTCGGTCGCCACTGGTGGAGAAGGTTTCCATAATTACCACCATGTCTTCCCCTGGGATTATCGAGCAGCGGAACTCGGTAACAATAGCCTCAATCTGACGACTAAATTCATAGATTTATTCGCAGCAATCGGATGGGCATATGATCTGAAGACGGTTTCGGAGGATATGATAAAACAAAGGATTAAACGCACTGGAGATGGAACGGATCTTTGGGGACACGAACAAAACTGTGATGAAGTGTGGGATGTAAAAGATAAATCAAGTTAA SEQ ID NO: 14mCherry C. tropicalis optimizedATGGTTTCTAAGGGTGAAGAAGACAACATGGCAATCATCAAGGAATTTATGCGTTTTAAGGTCCATATGGAAGGCTCCGTTAACGGCCACGAGTTCGAGATCGAGGGAGAAGGTGAGGGTAGACCATACGAAGGTACTCAAACCGCCAAGTTGAAAGTTACAAAGGGTGGTCCATTGCCATTTGCTTGGGATATCTTGTCCCCACAATTTATGTACGGATCAAAGGCATATGTCAAGCATCCTGCCGACATCCCAGATTACTTGAAGTTATCCTTTCCAGAAGGTTTTAAGTGGGAGAGAGTTATGAACTTTGAAGATGGCGGAGTTGTTACTGTTACTCAGGACTCTTCCTTGCAAGATGGTGAATTTATCTATAAAGTGAAATTGAGAGGTACTAACTTTCCATCCGACGGTCCAGTCATGCAAAAGAAGACAATGGGTTGGGAGGCTTCTTCCGAAAGAATGTACCCAGAAGACGGTGCATTGAAAGGTGAAATCAAGCAACGTTTAAAGTTGAAGGACGGTGGTCACTACGATGCCGAGGTCAAGACCACTTATAAGGCTAAGAAGCCAGTCCAATTGCCAGGTGCTTATAACGTTAACATCAAGTTAGATATTACTTCACACAACGAAGACTACACAATCGTTGAACAATATGAAAGAGCCGAAGGTAGACATTCTACCGGCGGCATGGACGAGTTATATAAGTAG SEQ ID NO: 15CaOLE1-A. segetum Z11 desaturaseATGACTACAGTTGAACAACTTGAAACTGTTGATATCACTAAATTGAATGCCATTGCTGCTGGTACTAATAAGAAGGTGCCAATGGCTCAAGGTGTCCAAACAACTACGATATTGAGGGAGGAAGAGCCGTCATTGACTTTCGTGGTACCTCAAGAACCGAGAAAGTATCAAATCGTGTACCCAAACCTTATCACATTTGGGTACTGGCATATAGCTGGTTTATACGGGCTATATTTGTGCTTTACTTCGGCAAAATGGCAAACAATTTTATTCAGTTTCATGCTCGTTGTGTTAGCAGAGTTGGGAATAACAGCCGGCGCTCACAGGTTATGGGCCCACAAAACATATAAAGCGAAGCTTCCCTTACAAATTATCTTAATGATATTAAACTCCATTGCCTTCCAAAATTCCGCCATTGATTGGGTGAGGGACCACCGTCTCCATCATAAGTACAGTGACACTGATGCAGACCCTCACAATGCTACTCGTGGTTTCTTCTATTCTCATGTTGGATGGTTGCTCGTAAGAAAACATCCAGAAGTCAAGAGACGTGGAAAGGAACTTGACATGTCTGATATTTACAACAATCCAGTGTTAAGATTTCAAAAGAAGTATGCTATACCCTTCATCGGGGCAATGTGCTTCGGATTACCAACTTTTATCCCTGTTTACTTCTGGGGAGAAACCTGGAGTAATGCTTGGCATATCACCATGCTTCGGTACATCCTCAACCTAAACATTACTTTCTTAGTCAACAGTGCTGCTCATATCTGGGGATACAAACCTTATGACATCAAAATATTGCCTGCCCAAAATATAGCAGTTTCCATAGTAACCGGCGGCGAAGTTTCCATAACTACCACCACGTTTTTTCCTTGGGATTATCGTGCAGCAGAATTGGGGAACAATTATCTTAATTTGACGACTAAGTTCATAGATTTCTTCGCTTGGATCGGATGGGCTTACGATCTTAAGACGGTGTCCAGTGATGTTATAAAAAGTAAGGCGGAAAGAACTGGTGATGGGACGAATCTTTGGGGTTTAGAAGACAAAGGTGAAGAAGATTTTTTGAAAATCTGGAAAGACAATTAA SEQ ID NO: 16A. segetum Z11 desaturaseATGGCTCAAGGTGTCCAAACAACTACGATATTGAGGGAGGAAGAGCCGTCATTGACTTTCGTGGTACCTCAAGAACCGAGAAAGTATCAAATCGTGTACCCAAACCTTATCACATTTGGGTACTGGCATATAGCTGGTTTATACGGGCTATATTTGTGCTTTACTTCGGCAAAATGGCAAACAATTTTATTCAGTTTCATGCTCGTTGTGTTAGCAGAGTTGGGAATAACAGCCGGCGCTCACAGGTTATGGGCCCACAAAACATATAAAGCGAAGCTTCCCTTACAAATTATCTTAATGATATTAAACTCCATTGCCTTCCAAAATTCCGCCATTGATTGGGTGAGGGACCACCGTCTCCATCATAAGTACAGTGACACTGATGCAGACCCTCACAATGCTACTCGTGGTTTCTTCTATTCTCATGTTGGATGGTTGCTCGTAAGAAAACATCCAGAAGTCAAGAGACGTGGAAAGGAACTTGACATGTCTGATATTTACAACAATCCAGTGTTAAGATTTCAAAAGAAGTATGCTATACCCTTCATCGGGGCAATGTGCTTCGGATTACCAACTTTTATCCCTGTTTACTTCTGGGGAGAAACCTGGAGTAATGCTTGGCATATCACCATGCTTCGGTACATCCTCAACCTAAACATTACTTTCTTAGTCAACAGTGCTGCTCATATCTGGGGATACAAACCTTATGACATCAAAATATTGCCTGCCCAAAATATAGCAGTTTCCATAGTAACCGGCGGCGAAGTTTCCATAACTACCACCACGTTTTTTCCTTGGGATTATCGTGCAGCAGAATTGGGGAACAATTATCTTAATTTGACGACTAAGTTCATAGATTTCTTCGCTTGGATCGGATGGGCTTACGATCTTAAGACGGTGTCCAGTGATGTTATAAAAAGTAAGGCGGAAAGAACTGGTGATGGGACGAATCTTTGGGGTTTAGAAGACAAAGGTGAAGAAGATTTTTTGAAAATCTGGAAAGACAATTAA SEQ ID NO: 17 A. transitella Z11 desaturaseATGGTCCCTAACAAGGGTTCCAGTGACGTTTTGTCTGAACATTCTGAGCCCCAGTTCACTAAACTCATAGCTCCACAAGCAGGGCCGAGGAAATACAAGATAGTGTATCGAAATTTGCTCACATTCGGCTATTGGCACTTATCAGCTGTTTATGGGCTCTACTTGTGCTTTACTTGTGCGAAATGGGCTACCATCTTATTTGCATTTTTCTTATACGTGATCGCGGAAATCGGTATAACAGGTGGCGCTCATAGGCTATGGGCACATCGGACTTATAAAGCCAAGTTGCCTTTAGAGATTTTGTTACTCATAATGAATTCTATTGCCTTCCAAGACACTGCTTTCACCTGGGCTCGAGATCACCGCCTTCATCACAAATATTCGGATACTGACGCTGATCCCCACAATGCTACCAGAGGGTTTTTCTATTCACATGTAGGCTGGCTTTTGGTGAAGAAACACCCTGAAGTCAAAGCAAGAGGAAAATACTTGTCGTTAGATGATCTTAAGAATAATCCATTGCTTAAATTCCAAAAGAAATACGCTATTCTAGTTATAGGCACGTTATGCTTCCTTATGCCAACATTTGTGCCCGTATACTTCTGGGGCGAGGGCATCAGCACGGCCTGGAACATCAATCTATTGCGATACGTCATGAATCTTAACATGACTTTCTTAGTTAACAGTGCAGCGCATATCTTTGGCAACAAACCATACGATAAGAGCATAGCCTCAGTCCAAAATATTTCAGTTAGCTTAGCTACTTTTGGCGAAGGATTCCATAATTACCATCACACTTACCCCTGGGATTATCGTGCGGCAGAATTAGGAAATAATAGGCTAAATATGACTACTGCTTTCATAGATTTCTTCGCTTGGATCGGCTGGGCTTATGACTTGAAGTCTGTGCCACAAGAGGCCATTGCAAAAAGGTGTGCGAAAACTGGCGATGGAACGGATATGTGGGGTCGAAAAAGATAA SEQ ID NO: 18T. ni Z11 desaturaseATGGCTGTGATGGCTCAAACAGTACAAGAAACGGCTACAGTGTTGGAAGAGGAAGCTCGCACAGTGACTCTTGTGGCTCCAAAGACAACGCCAAGGAAATATAAATATATATACACCAACTTTCTTACATTTTCATATGCGCATTTAGCTGCATTATACGGACTTTATTTGTGCTTCACCTCTGCGAAATGGGAAACATTGCTATTCTCTTTCGTACTCTTCCACATGTCAAATATAGGCATCACCGCAGGGGCTCACCGACTCTGGACTCACAAGACTTTCAAAGCCAAATTGCCTTTGGAAATTGTCCTCATGATATTCAACTCTTTAGCCTTTCAAAACACGGCTATTACATGGGCTAGAGAACATCGGCTACATCACAAATACAGCGATACTGATGCTGATCCCCACAATGCGTCAAGAGGGTTCTTCTACTCGCATGTTGGCTGGCTATTAGTAAAAAAACATCCCGATGTCTTAAAATATGGAAAAACTATAGACATGTCGGATGTATACAATAATCCTGTGTTAAAATTTCAGAAAAAGTACGCAGTACCCTTAATTGGAACAGTTTGTTTTGCTCTTCCAACTTTGATTCCAGTCTACTGTTGGGGCGAATCGTGGAACAACGCTTGGCACATAGCCTTATTTCGATACATATTCAATCTTAACGTGACTTTCCTAGTCAACAGTGCTGCGCATATCTGGGGGAATAAGCCTTATGATAAAAGCATCTTGCCCGCTCAAAACTTATTAGTTTCCTTCCTAGCAAGTGGAGAAGGCTTCCATAATTACCATCACGTCTTTCCATGGGATTACCGCACAGCAGAATTAGGGAATAACTTCTTAAATTTGACGACGTTATTCATTGATTTTTGTGCCTGGTTTGGATGGGCTTATGACTTGAAGTCTGTATCAGAGGATATTATAAAACAGAGAGCTAAACGAACAGGTGACGGTTCTTCAGGGGTCATTTGGGGATGGGACGACAAAGACATGGACCGCGATATAAAATCTAAAGCTAACATTTTTTATGCTAAAAAGG AATGASEQ ID NO: 19 H. zea Z11 desaturaseATGGCCCAAAGCTATCAATCAACTACGGTTTTGAGTGAGGAGAAAGAACTAACATTACAACATTTGGTGCCCCAAGCATCGCCCAGGAAGTATCAAATAGTGTATCCGAACCTCATTACGTTTGGTTACTGGCACATAGCCGGACTTTATGGCCTTTACTTGTGCTTCACTTCTGCTAAATGGGCTACGATTTTATTCAGCTACATCCTCTTCGTGTTAGCAGAAATAGGAATCACGGCTGGCGCTCACAGACTCTGGGCCCACAAAACTTACAAAGCGAAACTACCATTAGAAATACTCTTAATGGTATTCAACTCCATCGCTTTTCAAAACTCAGCCATTGACTGGGTGAGGGACCACCGACTCCACCATAAGTATAGCGATACAGATGCTGATCCCCACAATGCCAGCCGAGGGTTCTTTTATTCCCATGTAGGATGGCTACTTGTGAGAAAACATCCTGAAGTCAAAAAGCGAGGGAAAGAACTCAATATGTCCGATATTTACAACAATCCTGTCTTACGGTTTCAGAAAAAATACGCCATACCCTTCATTGGGGCTGTTTGTTTCGCCTTACCTACAATGATACCTGTTTACTTCTGGGGAGAAACCTGGTCCAATGCTTGGCATATCACCATGCTTCGCTACATCATGAACCTCAATGTCACCTTTTTGGTAAACAGCGCTGCTCATATATGGGGAAACAAGCCTTATGACGCAAAAATATTACCTGCACAAAATGTAGCTGTGTCGGTCGCCACTGGTGGAGAAGGTTTCCATAATTACCACCATGTCTTCCCCTGGGATTATCGAGCAGCGGAACTCGGTAACAATAGCCTCAATTTAACGACTAAATTCATAGATTTATTCGCAGCAATCGGATGGGCATATGATTTAAAGACGGTTTCGGAGGATATGATAAAACAAAGGATTAAACGCACTGGAGATGGAACGGATCTTTGGGGACACGAACAAAACTGTGATGAAGTGTGGGATGTAAAAGATAAATCAAGTTAA SEQ ID NO: 20 O. furnacalis Z9 desaturaseATGGCTCCTAATATTAAGGACGGAGCTGATTTGAACGGAGTTTTATTTGAAGATGACGCTAGCACCCCCGATTATGCCCTTGCCACGGCCCCAGTCCAGAAAGCAGACAACTATCCCAGAAAACTAGTGTGGAGAAACATCATACTCTTTGCATACCTTCACCTTGCCGCTGTGTATGGAGCATACCTATTCTTATTTTCAGCGAAATGGCAGACAGATATTTTTGCCTACATTCTTTACGTGATCTCAGGACTCGGCATCACAGCGGGAGCCCACCGCCTTTGGGCGCACAAGTCATACAAGGCTAAGTGGCCACTTAGACTCATTCTTATTATCTTCAACACTGTATCATTCCAGGACTCTGCTCTCGACTGGTCACGTGACCACCGCATGCACCACAAATACTCGGAGACCGACGCCGACCCGCACAACGCGACTCGAGGGTTCTTCTTCTCTCATATCGGCTGGTTATTAGTCCGCAAGCACCCGGAATTAAAGAGAAAGGGCAAGGGATTAGACTTAAGCGACTTGTATGCTGATCCCATCCTCCGTTTCCAGAAGAAGTACTATTTACTATTAATGCCTCTTGGCTGCTTCATCATGCCGACGGTGGTCCCGGTGTACTTCTGGGGTGAGACTTGGACTAACGCTTTCTTCGTCGCCGCGCTCTTCCGATACACCTTCATCCTCAATGTCACCTGGTTGGTCAACTCCGCCGCGCACAAGTGGGGCCACAAGCCCTATGACAGCAGCATCAAGCCTTCCGAGAACCTCTCAGTCTCCTTATTCGCGTTGGGCGAAGGATTCCACAACTACCACCACACATTCCCCTGGGACTACAAAACTGCCGAGCTCGGCAACAACAGACTCAATTTCACAACAAACTTCATCAACTTCTTCGCTAAAATCGGATGGGCTTACGACTTGAAAACGGTCTCCGACGAGATTATTCAGAATAGAGTCAAGCGCACAGGAGATGGCTCCCACCACTTATGGGGTTGGGGCGACAAGGATCAACCTAAAGAGGAGGTAAACGCAGCCATTAGAATTAATCCTAAAGACGAGTAA SEQ ID NO: 21 L. capitella Z9 desaturaseATGCCGCCGAACGTGACAGAGGCGAACGGAGTGTTATTTGAGAATGACGTGCAGACTCCTGACATGGGGCTAGAAGTGGCCCCTGTGCAGAAGGCTGACGAGCGTAAGATCCAGCTCGTTTGGAGGAACATCATCGCTTTTGCATGTCTTCATTTAGCAGCTGTGTATGGAGCTTATTTATTCTTCACCTCGGCTATATGGCAGACAGACATATTTGCATACATCCTTTACGTTATGTCTGGATTAGGAATCACGGCGGGAGCGCACAGATTATGGGCTCATAAGTCATACAAGGCGAAGTGGCCGTTAAGATTAATCCTCGTCGCATTCAACACTTTGGCATTCCAGGATTCGGCAATCGACTGGGCGCGCGACCACCGCATGCACCACAAGTACTCGGAGACGGATGCGGACCCACATAACGCCACTCGCGGCTTCTTCTTTTCGCACATTGGTTGGTTACTCTGCCGAAAACACCCGGAGCTAAAGCGCAAGGGCCAGGGCCTCGACTTAAGTGACCTCTACGCAGATCCTATTATTCGCTTCCAAAAGAAGTACTACTTATTGTTAATGCCGTTAGCCTGCTTTGTTCTTCCCACCATAATTCCGGTCTACCTCTGGGGCGAGTCCTGGAAAAACGCGTTCTTCGTAGCTGCAATGTTCCGTTACACGTTCATCCTCAACGTAACATGGCTCGTCAACTCCGCCGCCCACAAATGGGGAGGCAAGCCCTATGATAAGAACATCCAGCCCGCTCAGAACATCTCTGTAGCTATCTTCGCATTAGGCGAGGGCTTCCACAACTACCACCACACGTTCCCCTGGGACTACAAGACCGCTGAATTAGGAAACAACAGGTTAAATTTCACAACTTCGTTTATCAATTTCTTCGCAAGCTTCGGATGGGCCTACGACTTAAAGACCGTGTCGGACGAGATTATACAACAGCGCGTTAAGAGGACGGGAGATGGGAGCCATCACTTACGGGGCTGGGGCGACCAGGACATACCGGCCGAAGAAGCTCAAGCTGCTTTACGCATTAACC GTAAAGATGATTAGSEQ ID NO: 22 H. zea Z9 desaturaseATGGCTCCAAATATATCGGAGGATGTGAACGGGGTGCTCTTCGAGAGTGATGCAGCGACGCCGGACTTAGCGTTATCCACGCCGCCTGTGCAGAAGGCTGACAACAGGCCCAAGCAATTAGTGTGGAGGAACATACTATTATTCGCGTATCTTCACTTAGCGGCTCTTTACGGAGGTTATTTATTCCTCTTCTCAGCTAAATGGCAGACAGACATATTTGCCTACATCTTATATGTGATCTCCGGGCTTGGTATCACGGCTGGAGCACATCGCTTATGGGCCCACAAGTCCTACAAAGCTAAATGGCCTCTCCGAGTTATCTTAGTCATCTTTAACACAGTGGCATTCCAGGATGCCGCTATGGACTGGGCGCGCGACCACCGCATGCATCACAAGTACTCGGAAACCGATGCTGATCCTCATAATGCGACCCGAGGATTCTTCTTCTCTCACATTGGCTGGTTACTTGTCAGGAAACATCCCGACCTTAAGGAGAAGGGCAAGGGACTCGACATGAGCGACTTACTTGCTGACCCCATTCTCAGGTTCCAGAAAAAATACTACTTAATCTTAATGCCCTTGGCTTGCTTCGTGATGCCTACCGTGATTCCTGTGTACTTCTGGGGTGAAACCTGGACCAACGCATTCTTTGTGGCGGCCATGTTCCGCTACGCGTTCATCCTAAATGTGACGTGGCTCGTCAACTCTGCCGCTCACAAGTGGGGAGACAAGCCCTACGACAAAAGCATTAAGCCTTCCGAAAACTTGTCGGTCGCCATGTTCGCTCTCGGAGAAGGATTCCACAACTACCACCACACTTTCCCTTGGGACTACAAAACTGCTGAGTTAGGCAACAACAAACTCAACTTCACTACCACCTTTATTAACTTCTTCGCTAAAATTGGCTGGGCTTACGACTTAAAGACAGTGTCTGATGATATCGTCAAGAACAGGGTGAAGCGCACTGGTGACGGCTCCCACCACTTATGGGGCTGGGGAGACGAAAATCAATCCAAAGAAGAAATTGATGCCGCTATCAGAATCAATCCTAAGGACGATTAA SEQ ID NO: 23 T. pseudonana Z11 desaturaseATGGACTTTCTCTCCGGCGATCCTTTCCGGACACTCGTCCTTGCAGCACTTGTTGTCATCGGATTTGCTGCGGCGTGGCAATGCTTCTACCCGCCGAGCATCGTCGGCAAGCCTCGTACATTAAGCAATGGTAAACTCAATACCAGAATCCATGGCAAATTGTACGACCTCTCATCGTTTCAGCATCCAGGAGGCCCCGTGGCTCTTTCTCTTGTTCAAGGTCGCGACGGAACAGCTCTATTTGAGTCACACCATCCCTTCATACCTCGAAAGAATCTACTTCAGATCCTCTCCAAGTACGAGGTTCCGTCGACTGAAGACTCTGTTTCCTTCATCGCCACCCTAGACGAACTCAATGGTGAATCTCCGTACGATTGGAAGGACATTGAAAATGATGATTTCGTATCTGACCTACGAGCTCTCGTAATTGAGCACTTTTCTCCTCTCGCCAAGGAAAGGGGAGTTTCACTCGTTGAGTCGTCGAAGGCAACACCTCAGCGGTGGATGGTGGTTCTATTACTCCTTGCGTCGTTCTTCCTCAGCATCCCATTATATTTGAGTGGTTCGTGGACTTTCGTTGTCGTCACTCCCATCCTCGCTTGGTTAGCGGTTGTCAATTACTGGCACGATGCTACTCACTTTGCATTGAGCAGCAACTGGATTTTGAATGCTGCGCTCCCATATCTCCTCCCTCTCCTATCGAGTCCGTCAATGTGGTATCATCATCACGTCATTGGACATCACGCATACACCAACATTTCCAAAAGAGATCCAGATCTTGCTCACGCTCCACAACTCATGAGAGAACACAAGAGTATCAAATGGAGACCATCTCACTTAAATCAAACACAGCTTCCGCGGATTCTCTTCATCTGGTCGATTGCAGTCGGTATTGGGTTGAACTTATTAAACGACGTGAGAGCACTAACCAAGCTTTCATACAACAACGTTGTTCGGGTGGAGAAGATGTCATCGTCGCGAACATTACTCCATTTCCTTGGACGTATGTTGCACATCTTTGTGACTACACTTTGGCCCTTTTTGGCGTTTCCGGTGTGGAAGGCCATCGTTTGGGCGACTGTACCGAATGCCATATTAAGTTTGTGCTTCATGTTAAATACGCAAATCAATCACCTCATCAACACGTGTGCACATGCTTCCGATAACAACTTTTACAAGCATCAAGTTGTAACTGCTCAGAACTTTGGCCGATCAAGTGCCTTTTGCTTCATCTTCTCGGGAGGTCTCAACTACCAAATTGAACATCATTTGTTGCCGACGGTGAACCATTGCCATTTGCCAGCTTTGGCCCCGGGTGTAGAGCGTTTGTGTAAGAAACACGGGGTGACATACAACTCTGTTGAAGGATACAGAGAGGCCATCATTGCACACTTTGCACATACCAAAGATATGTCGACGAAGCCTACTGA TTGASEQ ID NO: 24 Native T. ni Z11 desaturaseATGGCTGTGATGGCTCAAACAGTACAAGAAACGGCTACAGTGTTGGAAGAGGAAGCTCGCACAGTGACTCTTGTGGCTCCAAAGACAACGCCAAGGAAATATAAATATATATACACCAACTTTCTTACATTTTCATATGCGCATTTAGCTGCATTATACGGACTTTATTTGTGCTTCACCTCTGCGAAATGGGAAACATTGCTATTCTCTTTCGTACTCTTCCACATGTCAAATATAGGCATCACCGCAGGGGCTCACCGACTCTGGACTCACAAGACTTTCAAAGCCAAATTGCCTTTGGAAATTGTCCTCATGATATTCAACTCTTTAGCCTTTCAAAACACGGCTATTACATGGGCTAGAGAACATCGGCTACATCACAAATACAGCGATACTGATGCTGATCCCCACAATGCGTCAAGAGGGTTCTTCTACTCGCATGTTGGCTGGCTATTAGTAAAAAAACATCCCGATGTCCTGAAATATGGAAAAACTATAGACATGTCGGATGTATACAATAATCCTGTGTTAAAATTTCAGAAAAAGTACGCAGTACCCTTAATTGGAACAGTTTGTTTTGCTCTGCCAACTTTGATTCCAGTCTACTGTTGGGGCGAATCGTGGAACAACGCTTGGCACATAGCCTTATTTCGATACATATTCAATCTTAACGTGACTTTCCTAGTCAACAGTGCTGCGCATATCTGGGGGAATAAGCCTTATGATAAAAGCATCTTGCCCGCTCAAAACCTGCTGGTTTCCTTCCTAGCAAGTGGAGAAGGCTTCCATAATTACCATCACGTCTTTCCATGGGATTACCGCACAGCAGAATTAGGGAATAACTTCCTGAATTTGACGACGCTGTTCATTGATTTTTGTGCCTGGTTTGGATGGGCTTATGACTTGAAGTCTGTATCAGAGGATATTATAAAACAGAGAGCTAAACGAACAGGTGACGGTTCTTCAGGGGTCATTTGGGGATGGGACGACAAAGACATGGACCGCGATATAAAATCTAAAGCTAACATTTTTTATGCTAAAAAGG AATGASEQ ID NO: 25 H. zea Z11 desaturaseATGGCCCAAAGCTATCAATCAACTACGGTTTTGAGTGAGGAGAAAGAACTAACACTGCAACATTTGGTGCCCCAAGCATCGCCCAGGAAGTATCAAATAGTGTATCCGAACCTCATTACGTTTGGTTACTGGCACATAGCCGGACTTTATGGCCTTTACTTGTGCTTCACTTCTGCTAAATGGGCTACGATTTTATTCAGCTACATCCTCTTCGTGTTAGCAGAAATAGGAATCACGGCTGGCGCTCACAGACTCTGGGCCCACAAAACTTACAAAGCGAAACTACCATTAGAAATACTCTTAATGGTATTCAACTCCATCGCTTTTCAAAACTCAGCCATTGACTGGGTGAGGGACCACCGACTCCACCATAAGTATAGCGATACAGATGCTGATCCCCACAATGCCAGCCGAGGGTTCTTTTATTCCCATGTAGGATGGCTACTTGTGAGAAAACATCCTGAAGTCAAAAAGCGAGGGAAAGAACTCAATATGTCCGATATTTACAACAATCCTGTCCTGCGGTTTCAGAAAAAATACGCCATACCCTTCATTGGGGCTGTTTGTTTCGCCTTACCTACAATGATACCTGTTTACTTCTGGGGAGAAACCTGGTCCAATGCTTGGCATATCACCATGCTTCGCTACATCATGAACCTCAATGTCACCTTTTTGGTAAACAGCGCTGCTCATATATGGGGAAACAAGCCTTATGACGCAAAAATATTACCTGCACAAAATGTAGCTGTGTCGGTCGCCACTGGTGGAGAAGGTTTCCATAATTACCACCATGTCTTCCCCTGGGATTATCGAGCAGCGGAACTCGGTAACAATAGCCTCAATCTGACGACTAAATTCATAGATTTATTCGCAGCAATCGGATGGGCATATGATCTGAAGACGGTTTCGGAGGATATGATAAAACAAAGGATTAAACGCACTGGAGATGGAACGGATCTTTGGGGACACGAACAAAACTGTGATGAAGTGTGGGATGTAAAAGATAAATCAAGTTAA SEQ ID NO: 26T. ni Z11 desaturase Homo sapiens optimizedATGGCCGTGATGGCCCAGACCGTGCAGGAGACCGCAACAGTGCTGGAGGAGGAGGCAAGGACCGTGACACTGGTGGCACCCAAGACCACACCTAGAAAGTACAAGTATATCTACACCAACTTCCTGACCTTCAGCTACGCACACCTGGCCGCCCTGTATGGACTGTACCTGTGCTTTACCTCCGCCAAGTGGGAGACACTGCTGTTCTCTTTTGTGCTGTTCCACATGAGCAATATCGGAATCACCGCAGGAGCACACAGGCTGTGGACCCACAAGACATTCAAGGCCAAGCTGCCTCTGGAGATCGTGCTGATGATCTTCAACTCTCTGGCCTTTCAGAATACCGCCATCACATGGGCCCGGGAGCACAGACTGCACCACAAGTATAGCGACACCGATGCAGACCCACACAACGCAAGCAGGGGCTTCTTTTACTCCCACGTGGGCTGGCTGCTGGTGAAGAAGCACCCCGACGTGCTGAAGTATGGCAAGACAATCGACATGTCCGACGTGTACAACAATCCCGTGCTGAAGTTTCAGAAGAAGTATGCCGTGCCTCTGATCGGCACCGTGTGCTTCGCCCTGCCAACACTGATCCCCGTGTATTGTTGGGGCGAGTCTTGGAACAATGCCTGGCACATCGCCCTGTTCCGGTACATCTTTAACCTGAATGTGACCTTTCTGGTGAACTCCGCCGCCCACATCTGGGGCAATAAGCCTTACGACAAGTCTATCCTGCCAGCCCAGAACCTGCTGGTGTCCTTCCTGGCCTCTGGCGAGGGCTTTCACAATTATCACCACGTGTTCCCATGGGACTACAGGACCGCAGAGCTGGGCAACAATTTTCTGAACCTGACCACACTGTTCATCGATTTTTGTGCCTGGTTCGGCTGGGCCTATGACCTGAAGTCTGTGAGCGAGGATATCATCAAGCAGAGGGCAAAGAGGACAGGCGATGGCAGCTCCGGCGTGATCTGGGGATGGGACGATAAGGATATGGACAGAGATATCAAGAGCAAGGCCAATATCTTCTACGCCAAGAAGG AGTGASEQ ID NO: 27 H. zea Z11 desaturase Homo sapiens optimizedATGGCACAGTCATATCAGAGCACTACCGTCCTGAGCGAAGAGAAGGAACTGACACTGCAGCACCTGGTCCCACAGGCATCACCTAGAAAGTACCAGATCGTGTATCCAAACCTGATCACCTTCGGCTACTGGCACATCGCCGGCCTGTACGGCCTGTATCTGTGCTTTACCTCCGCCAAGTGGGCCACAATCCTGTTCTCTTACATCCTGTTTGTGCTGGCAGAGATCGGAATCACCGCAGGAGCACACAGACTGTGGGCACACAAGACATATAAGGCCAAGCTGCCCCTGGAGATCCTGCTGATGGTGTTCAACAGCATCGCCTTTCAGAATTCCGCCATCGATTGGGTGCGGGACCACAGACTGCACCACAAGTACTCCGACACCGATGCCGACCCCCACAACGCCTCTAGGGGCTTCTTTTATAGCCACGTGGGATGGCTGCTGGTGCGGAAGCACCCTGAGGTGAAGAAGAGAGGCAAGGAGCTGAATATGTCTGATATCTACAACAATCCTGTGCTGCGCTTCCAGAAGAAGTATGCCATCCCATTCATCGGCGCCGTGTGCTTTGCCCTGCCCACCATGATCCCCGTGTACTTTTGGGGCGAGACATGGAGCAACGCCTGGCACATCACAATGCTGCGGTATATCATGAACCTGAATGTGACATTCCTGGTGAACTCCGCCGCCCACATCTGGGGCAATAAGCCATACGACGCCAAGATCCTGCCCGCCCAGAACGTGGCCGTGAGCGTGGCAACCGGAGGAGAGGGCTTCCACAATTACCACCACGTGTTTCCTTGGGATTATCGGGCCGCCGAGCTGGGCAACAATTCTCTGAATCTGACCACAAAGTTCATCGACCTGTTTGCCGCCATCGGCTGGGCCTATGATCTGAAGACAGTGAGCGAGGACATGATCAAGCAGAGGATCAAGCGCACCGGCGATGGCACAGACCTGTGGGGGCACGAGCAGAACTGTGATGAAGTGTGGGATGTGAAAGACAAGTCCTCCTAA SEQ ID NO: 28Y. lipolytica OLE1 leader - T. ni Z11 desaturase Homo sapiens optimizedATGGTGAAGAACGTGGACCAGGTGGATCTGTCTCAGGTGGACACCATCGCAAGCGGAAGGGATGTGAATTATAAGGTGAAGTACACATCTGGCGTGAAGACCACACCAAGAAAGTACAAGTATATCTACACCAACTTCCTGACATTTTCTTACGCCCACCTGGCCGCCCTGTATGGCCTGTACCTGTGCTTTACCAGCGCCAAGTGGGAGACACTGCTGTTCTCCTTTGTGCTGTTCCACATGTCTAATATCGGAATCACCGCAGGAGCACACAGGCTGTGGACCCACAAGACATTCAAGGCCAAGCTGCCCCTGGAGATCGTGCTGATGATCTTCAACTCCCTGGCCTTTCAGAATACCGCCATCACATGGGCCCGGGAGCACAGACTGCACCACAAGTATTCTGACACCGATGCAGACCCACACAACGCAAGCAGGGGCTTCTTTTACTCCCACGTGGGCTGGCTGCTGGTGAAGAAGCACCCTGACGTGCTGAAGTATGGCAAGACAATCGACATGAGCGACGTGTACAACAATCCTGTGCTGAAGTTTCAGAAGAAGTATGCCGTGCCACTGATCGGCACCGTGTGCTTCGCCCTGCCCACACTGATCCCCGTGTACTGTTGGGGCGAGTCCTGGAACAATGCCTGGCACATCGCCCTGTTCCGGTACATCTTTAACCTGAATGTGACCTTTCTGGTGAACAGCGCCGCCCACATCTGGGGCAATAAGCCATACGACAAGTCCATCCTGCCCGCCCAGAACCTGCTGGTGTCCTTCCTGGCCTCTGGCGAGGGCTTTCACAATTATCACCACGTGTTCCCTTGGGACTACAGGACCGCAGAGCTGGGCAACAATTTTCTGAACCTGACCACACTGTTCATCGATTTTTGTGCCTGGTTCGGCTGGGCCTATGACCTGAAGTCTGTGAGCGAGGATATCATCAAGCAGAGGGCAAAGAGGACAGGCGATGGCAGCTCCGGCGTGATCTGGGGATGGGACGATAAGGATATGGACAGAGATATCAAGTCCAAGGCCAATATCTTCTACGCCAAGAAGGAGTGA SEQ ID NO: 29Y. lipolytica OLE1 leader - H. zea Z11 desaturase Homo sapiens optimizedATGGTGAAAAACGTGGACCAAGTGGATCTCTCGCAGGTCGACACCATTGCCTCCGGCCGAGATGTCAACTACAAGGTCAAGTACACCTCCGGCGTTCGCAAGTATCAGATCGTGTATCCTAACCTGATCACCTTCGGCTACTGGCATATCGCTGGACTGTACGGACTGTATCTGTGCTTCACTTCCGCCAAGTGGGCCACCATCCTGTTCTCTTACATCCTGTTTGTGCTGGCAGAGATCGGAATCACCGCAGGAGCACACAGACTGTGGGCACACAAGACATATAAGGCCAAGCTGCCACTGGAGATCCTGCTGATGGTGTTCAACAGCATCGCCTTTCAGAATTCCGCCATCGATTGGGTGCGGGACCACAGACTGCACCACAAGTACTCCGACACAGATGCCGACCCCCACAACGCCTCTAGGGGCTTCTTTTATAGCCACGTGGGATGGCTGCTGGTGCGGAAGCACCCTGAGGTGAAGAAGAGAGGCAAGGAGCTGAATATGTCTGATATCTACAACAATCCTGTGCTGCGCTTCCAGAAGAAGTATGCCATCCCATTCATCGGCGCCGTGTGCTTTGCCCTGCCCACCATGATCCCCGTGTACTTTTGGGGCGAGACATGGAGCAACGCCTGGCACATCACAATGCTGCGGTATATCATGAACCTGAATGTGACATTCCTGGTGAACTCCGCCGCCCACATCTGGGGCAATAAGCCATACGACGCCAAGATCCTGCCCGCCCAGAACGTGGCCGTGAGCGTGGCAACCGGAGGAGAGGGCTTCCACAATTACCACCACGTGTTTCCATGGGATTATAGGGCAGCAGAGCTGGGAAACAATTCTCTGAATCTGACCACAAAGTTCATCGACCTGTTTGCCGCCATCGGCTGGGCCTATGATCTGAAGACAGTGAGCGAGGACATGATCAAGCAGAGGATCAAGCGCACCGGCGATGGCACAGACCTGTGGGGGCACGAGCAGAATTGTGATGAAGTGTGGGATGTGAAGGATAAAAGCAGTTGA SEQ ID NO: 30Native A. transitella Z11 desaturaseATGGTCCCTAACAAGGGTTCCAGTGACGTTTTGTCTGAACATTCTGAGCCCCAGTTCACTAAACTCATAGCTCCACAAGCAGGGCCGAGGAAATACAAGATAGTGTATCGAAATTTGCTCACATTCGGCTATTGGCACTTATCAGCTGTTTATGGGCTCTACTTGTGCTTTACTTGTGCGAAATGGGCTACCATCTTATTTGCATTTTTCTTATACGTGATCGCGGAAATCGGTATAACAGGTGGCGCTCATAGGCTATGGGCACATCGGACTTATAAAGCCAAGTTGCCTTTAGAGATTTTGTTACTCATAATGAATTCTATTGCCTTCCAAGACACTGCTTTCACCTGGGCTCGAGATCACCGCCTTCATCACAAATATTCGGATACTGACGCTGATCCCCACAATGCTACCAGAGGGTTTTTCTATTCACATGTAGGCTGGCTTTTGGTGAAGAAACACCCTGAAGTCAAAGCAAGAGGAAAATACTTGTCGTTAGATGATCTTAAGAATAATCCATTGCTTAAATTCCAAAAGAAATACGCTATTCTAGTTATAGGCACGTTATGCTTCCTTATGCCAACATTTGTGCCCGTATACTTCTGGGGCGAGGGCATCAGCACGGCCTGGAACATCAATCTATTGCGATACGTCATGAATCTTAACATGACTTTCTTAGTTAACAGTGCAGCGCATATCTTTGGCAACAAACCATACGATAAGAGCATAGCCTCAGTCCAAAATATTTCAGTTAGCTTAGCTACTTTTGGCGAAGGATTCCATAATTACCATCACACTTACCCCTGGGATTATCGTGCGGCAGAATTAGGAAATAATAGGCTAAATATGACTACTGCTTTCATAGATTTCTTCGCTTGGATCGGCTGGGCTTATGACTTGAAGTCTGTGCCACAAGAGGCCATTGCAAAAAGGTGTGCGAAAACTGGCGATGGAACGGATATGTGGGGTCGAAAAAGATAA SEQ ID NO: 31pPV0228_-_Z11 Helicoverpa zea desaturaseATGGCCCAAAGCTATCAATCAACTACGGTTTTGAGTGAGGAGAAAGAACTAACATTACAACATTTGGTGCCCCAAGCATCGCCCAGGAAGTATCAAATAGTGTATCCGAACCTCATTACGTTTGGTTACTGGCACATAGCCGGACTTTATGGCCTTTACTTGTGCTTCACTTCTGCTAAATGGGCTACGATTTTATTCAGCTACATCCTCTTCGTGTTAGCAGAAATAGGAATCACGGCTGGCGCTCACAGACTCTGGGCCCACAAAACTTACAAAGCGAAACTACCATTAGAAATACTCTTAATGGTATTCAACTCCATCGCTTTTCAAAACTCAGCCATTGACTGGGTGAGGGACCACCGACTCCACCATAAGTATAGCGATACAGATGCTGATCCCCACAATGCCAGCCGAGGGTTCTTTTATTCCCATGTAGGATGGCTACTTGTGAGAAAACATCCTGAAGTCAAAAAGCGAGGGAAAGAACTCAATATGTCCGATATTTACAACAATCCTGTCTTACGGTTTCAGAAAAAATACGCCATACCCTTCATTGGGGCTGTTTGTTTCGCCTTACCTACAATGATACCTGTTTACTTCTGGGGAGAAACCTGGTCCAATGCTTGGCATATCACCATGCTTCGCTACATCATGAACCTCAATGTCACCTTTTTGGTAAACAGCGCTGCTCATATATGGGGAAACAAGCCTTATGACGCAAAAATATTACCTGCACAAAATGTAGCTGTGTCGGTCGCCACTGGTGGAGAAGGTTTCCATAATTACCACCATGTCTTCCCCTGGGATTATCGAGCAGCGGAACTCGGTAACAATAGCCTCAATTTAACGACTAAATTCATAGATTTATTCGCAGCAATCGGATGGGCATATGATTTAAAGACGGTTTCGGAGGATATGATAAAACAAAGGATTAAACGCACTGGAGATGGAACGGATCTTTGGGGACACGAACAAAACTGTGATGAAGTGTGGGATGTAAAAGATAAATCAAGTTAA SEQ ID NO: 32pPV0228_-_Helicoverpa armigera reductase codon optimizedATGGTCGTTTTAACTTCTAAAGAGACAAAACCTTCAGTAGCTGAGTTTTATGCGGGAAAATCTGTTTTTATTACGGGTGGCACTGGATTCCTTGGAAAGGTATTCATAGAGAAACTTTTATATAGCTGTCCAGATATCGAGAATATCTACATGCTCATACGAGAGAAGAAAGGTCTTTCTGTTAGCGAAAGAATAAAACAGTTCCTTGATGACCCGCTCTTTACCAGACTAAAAGACAAAAGACCAGCTGACTTAGAGAAGATTGTATTAATACCAGGAGATATTACTGCTCCTGACTTAGGCATTAATTCTGAAAACGAGAAGATGCTTATAGAGAAGGTATCGGTGATTATTCATTCGGCTGCTACGGTGAAGTTTAATGAGCCTCTCCCTACGGCTTGGAAGATCAACGTGGAAGGAACCAGAATGATGTTAGCTTTGAGTCGAAGAATGAAGCGGATTGAGGTTTTCATTCACATATCGACAGCATACACGAACACAAACAGGGAAGTGGTTGACGAGATCTTATACCCAGCTCCTGCTGATATCGACCAAGTTCATCAGTATGTCAAAGATGGAATCTCTGAGGAAGACACTGAGAAAATATTAAATGGTCGTCCAAATACGTACACGTTTACGAAAGCGTTAACTGAGCATTTAGTTGCTGAGAACCAAGCCTACGTACCCACTATTATCGTCAGGCCGTCAGTCGTGGCAGCAATAAAAGATGAGCCATTAAAAGGTTGGTTAGGCAACTGGTTTGGAGCGACTGGTCTCACCGTGTTCACCGCTAAGGGTCTCAACCGAGTCATCTACGGTCATTCTAGCTACATCGTAGACTTAATTCCTGTGGATTATGTCGCTAATTTAGTGATTGCTGCTGGGGCTAAGAGTAGCAAGTCAACTGAGTTGAAGGTATACAACTGCTGCAGCAGCTCCTGCAATCCCGTCACTATTGGCACGTTAATGAGCATGTTTGCTGACGATGCCATCAAACAGAAGTCGTATGCTATGCCGCTACCGGGGTGGTACATATTCACGAAATATAAGTGGTTAGTTCTTCTTTTAACATTTCTCTTCCAAGTTATACCGGCGTATGTCACAGATCTCTCCAGGCACTTGATTGGGAAGAGTCCACGGTACATAAAACTCCAATCACTAGTAAATCAAACGCGCTCTTCAATCGACTTCTTCACGAATCACTCCTGGGTGATGAAGGCAGACAGAGTGAGAGAGTTATATGCGTCTCTTTCCCCCGCAGACAAGTACTTATTTCCCTGTGATCCTACGGACATTAACTGGACACATTACATACAAGACTACTGTTGGGGAGTCCGACATTTTTTGGAGAAAAAAAGCTACGAATAA SEQ ID NO: 33pPV0228_-_ICL_promoterTATTAGGCGAAGAGGCATCTAGTAGTAGTGGCAGTGGTGAGAACGTGGGCGCTGCTATAGTGAACAATCTCCAGTCGATGGTTAAGAAGAAGAGTGACAAACCAGCAGTGAATGACTTGTCTGGGTCCGTGAGGAAAAGAAAGAAGCCCGACACAAAGGACAGTAACGTCAAGAAACCCAAGAAATAGGGGGGACCTGTTTAGATGTATAGGAATAAAAACTCCGAGATGATCTCAATGTGTAATGGAGTTGTAATATTGCAAAGGGGGAAAATCAAGACTCAAACGTGTGTATGAGTGAGCGTACGTATATCTCCGAGAGTAGTATGACATAATGATGACTGTGAATCATCGTAATCTCACACAAAAACCCCATTGTCGGCCATATACCACACCAAGCAACACCACATATCCCCCGGAAAAAAAAACGTGAAAAAAAGAAACAATCAAAACTACAACCTACTCCTTGATCACACAGTCATTGATCAAGTTACAGTTCCTGCTAGGGAATGACCAAGGTACAAATCAGCACCTTAATGGTTAGCACGCTCTCTTACTCTCTCTCACAGTCTTCCGGCCCCTATTCAAAATTCTGCACTTCCATTTGACCCCAGGGTTGGGAAACAGGGCCACAAAAGAAAAACCCGACGTGAATGAAAAAACTAAGAAAAGAAAAAAAATTATCACACCAGAAATTTACCTAATTGGGTAATTCCCATCGGTGTTTTTCCTGGATTGTCGCACGCACGCATGCTGAAAAAAGTGTTCGAGTTTTGCTTTTGCCTCGGAGTTTCACGCAAGTTTTTCGATCTCGGAACCGGAGGGCGGTCGCCTTGTTGTTTGTGATGTCGTGCTTTGGGTGTTCTAATGTGCTGTTATTGTGCTCTTTTTTTTTCTTCTTTTTTTGGTGATCATATGATATTGCTCGGTAGATTACTTTCGTGTGTAGGTATTCTTTTAGACGTTTGGTTATTGGGTAGATATGAGAGAGAGAGAGTGGGTGGGGGAGGAGTTGGTTGTAGGAGGGACCCCTGGGAGGAAGTGTAGTTGAGTTTTCCCTGACGAATGAAAATACGTTTTTGAGAAGATAATACAGGAAAGGTGTGTCGGTGAATTTCCATCTATCCGAGGATATGAGTGGAGGAGAGTCGTGTGCGTGTGGTTAATTTAGGATCAGTGGAACACACAAAGTAACTAAGACAGAGAGACAGAGAGAAAAATCTGGGGAAGAGACAAAGAGTCAGAGTGTGTGAGTTATTCTGTATTGTGAAATTTTTTTGCCCAACTACATAATATTGCTGAAACTAATTTTACTTAAAAAGAAAAGCCAACAACGTCCCCAGTAAAACTTTTCTATAAATATCAGCAGTTTTCCCTTTCCTCCATTCCTCTTCTTGTCTTTTTTCTTACTTTCCCTTTTTTATACCTTTTCATTATCATCCTTTATAATTGTCTAACCAACAACTATATATCTATCAA SEQ ID NO: 34pPV0228_-_TEF_Candida tropicalis_promoter_regionAGGAAGACAACCAAAAGAAAGATCAAATTGACTAAATGTTGAACAGACCAAAAAAAAAGAACAACAAATAGATAAATTACAACATATTAATCTTTTGATATGTTGTTGAATATTCTAGTAAATCTAATGATCTCAATAGTGGTTATCATTCACTCTCTTCGTCCTCCTCTCTCCCCTCCTCCTCTTGCAGTATATTAAGCAATAAAAAAAAAAAAAAAAAAAGAAAATCTGCCAACACACACAAAAAAAACTTACATAGTCGTGTACCAGTGTCAATATTTCACCAGCGCAGAGAAAAGAAGATGAACAGAAAAATTTTCTCTTTGGTTTTGTCTTTGGTTTTGTATTAATCTCATTGAAAAATTTTTTCTCTCTCTCTCTCTCTCTCTCTCACTCACACACTCACTCGCATTTCGTTTGGGTTACAGCAGAAGTCAGACAGAAAAAAAAAATCGTATATAACTCTCATCAAATGCCCTAGAGAAAAATTTTTCTTCTATCCTTTTTTTTTTCTTCTTCTTCTTCTTTTCCTTTTTTCTTTTAGAAGATCTTTTTGAATTCATCAAAGATATATATTTAATCAATC SEQ ID NO: 35pPV0228_-_ICL_terminatorAAGAAAAAAGAAAAGGTAAAGAACTTCATTTGAGATGAACTTTTGTATATGACTTTTAGTTTCTACTTTTTTTTTTATTTATTGCTTAATTTTCTTTATTTCAATCCCCCATAGTTTGTGTAGAATATATTTATTCATTCTGGTAACTCAAACACGTAGCAAGCTCGTTGCATCTCGCCTCGTCACGGGTACAGCTCTGGAACCAAGACAAAGTTGATCCGAACCCTCTCGCTATTCCTTGCTATGCTATCCACGAGATGGGGTTTATCAGCCCAGGCAAGTCACTAAA SEQ ID NO: 36 pPV0228_-_TEF_terminatorGCTGATTAATGAATAATTAATAAGTATTGTTTTTTTTGTTTTTAATATATATATATCTTGAAATTAGTATAAAAAAAATCTTTTTTTTTTCTTTTTTATTTATTTTATCAATAGTTTATATATATATATATATAAACTTGTAAGAGATTAGGTATATCTAAEAGTGATACTACTAATAGTGCTTAATATCTTTGTTAAACAAGAAAATAAAATAAAC SEQ ID NO: 37SapI-tLIP2-pEXP1-HA_FAR-SapI (insert into pPV199 creating pPV247)GCCTGAAGAGCGCTATTTATCACTCTTTACAACTTCTACCTCAACTATCTACTTTAATAAATGAATATCGTTTATTCTCTATGATTACTGTATATGCGTTCCTCCATGGGAGTTTGGCGCCCGTTTTTTCGAGCCCCACACGTTTCGGTGAGTATGAGCGGCGGCAGATTCGAGCGTTTCCGGTTTCCGCGGCTGGACGAGAGCCCATGATGGGGGCTCCCACCACCAGCAATCAGGGCCCTGATTACACACCCACCTGTAATGTCATGCTGTTCATCGTGGTTAATGCTGCTGTGTGCTGTGTGTGTGTGTTGTTTGGCGCTCATTGTTGCGTTATGCAGCGTACACCACAATATTGGAAGCTTATTAGCCTTTCTATTTTTTCGTTTGCAAGGCTTAACAACATTGCTGTGGAGAGGGATGGGGATATGGAGGCCGCTGGAGGGAGTCGGAGAGGCGTTTTGGAGCGGCTTGGCCTGGCGCCCAGCTCGCGAAACGCACCTAGGACCCTTTGGCACGCCGAAATGTGCCACTTTTCAGTCTAGTAACGCCTTACCTACGTCATTCCATGCATGCATGTTTGCGCCTTTTTTCCCTTGCCCTTGATCGCCACACAGTACAGTGCACTGTACAGTGGAGGTTTTGGGGGGGTCTTAGATGGGAGCTAAAAGCGGCCTAGCGGTACACTAGTGGGATTGTATGGAGTGGCATGGAGCCTAGGTGGAGCCTGACAGGACGCACGACCGGCTAGCCCGTGACAGACGATGGGTGGCTCCTGTTGTCCACCGCGTACAAATGTTTGGGCCAAAGTCTTGTCAGCCTTGCTTGCGAACCTAATTCCCAATTTTGTCACTTCGCACCCCCATTGATCGAGCCCTAACCCCTGCCCATCAGGCAATCCAATTAAGCTCGCATTGTCTGCCTTGTTTAGTTTGGCTCCTGCCCGTTTCGGCGTCCACTTGCACAAACACAAACAAGCATTATATATAAGGCTCGTCTCTCCCTCCCAACCACACTCACTTTTTTGCCCGTCTTCCCTTGCTAACACAAAAGTCAAGAACACAAACAACCACCCCAACCCCCTTACACACAAGACATATCTACAGCAATGGTGGTGCTGACCAGCAAGGAGACAAAGCCTTCCGTGGCCGAGTTCTACGCCGGCAAGTCCGTGTTTATCACAGGCGGCACCGGCTTCCTGGGCAAGGTGTTTATCGAGAAGCTGCTGTACTCTTGCCCAGACATCGAGAACATCTATATGCTGATCCGGGAGAAGAAGGGCCTGAGCGTGTCCGAGAGAATCAAGCAGTTCCTGGACGATCCCCTGTTTACACGGCTGAAGGACAAGAGACCTGCCGATCTGGAGAAGATCGTGCTGATCCCAGGCGACATCACCGCACCAGATCTGGGCATCAACTCCGAGAATGAGAAGATGCTGATCGAGAAGGTGTCCGTGATCATCCACTCTGCCGCCACCGTGAAGTTCAACGAGCCCCTGCCTACAGCCTGGAAGATCAATGTGGAGGGCACCAGGATGATGCTGGCCCTGAGCCGGAGAATGAAGCGCATCGAGGTGTTTATCCACATCTCCACAGCCTACACCAACACAAATCGGGAGGTGGTGGACGAGATCCTGTACCCAGCCCCCGCCGACATCGATCAGGTGCACCAGTATGTGAAGGACGGCATCAGCGAGGAGGATACCGAGAAGATCCTGAACGGCCGGCCAAATACCTACACATTCACCAAGGCCCTGACAGAGCACCTGGTGGCCGAGAACCAGGCCTATGTGCCTACCATCATCGTGAGACCATCCGTGGTGGCCGCCATCAAGGATGAGCCCCTGAAGGGATGGCTGGGAAACTGGTTCGGAGCAACAGGACTGACCGTGTTTACAGCCAAGGGCCTGAATAGAGTGATCTACGGCCACAGCTCCTATATCGTGGACCTGATCCCCGTGGATTACGTGGCAAACCTGGTCATCGCAGCAGGAGCCAAGTCTAGCAAGTCTACCGAGCTGAAGGTGTATAACTGCTGTTCCTCTAGCTGTAATCCTGTGACCATCGGCACACTGATGTCCATGTTCGCCGACGATGCCATCAAGCAGAAGTCTTACGCCATGCCTCTGCCAGGCTGGTACATCTTTACAAAGTATAAGTGGCTGGTGCTGCTGCTGACCTTCCTGTTTCAGGTCATCCCAGCCTACGTGACCGATCTGTCTAGGCACCTGATCGGCAAGAGCCCCCGCTATATCAAGCTGCAGTCTCTGGTGAACCAGACCAGGTCCTCTATCGACTTCTTTACAAATCACAGCTGGGTCATGAAGGCCGATAGGGTGCGCGAGCTGTACGCCTCTCTGAGCCCTGCCGACAAGTATCTGTTCCCCTGCGACCCTACCGATATCAATTGGACACACTACATCCAGGATTATTGTTGGGGCGTGCGCCACTTCCTGGAGAAGAAGTCCTATGAGTGAGCCTGAAGAGC SEQ ID NO: 38NcoI-pTAL-AleI (insert into pPV247 creating pPV248)CCATGGGTAAGCAGGTGGCTCCGTTTGTGTCTTTGTGTTTTTCCCCTCCTTTTTGGACCATTTGTCAGCATGTTGCGTAGGTCTGGGTGTTTGACTGTTCAGGTGGTGGATGACGGATGCATCATCTGACGGCAGAGTGGGTACCTGGCAGTGGCAGGCTCGCAGACGAGGTAGAGAGATTCTGAAAGGAGCCATTGACAGATGGAGAATTGGATACTCCTGGTATGTCCTCCGTTTCCACTTTTGACGTTGGTGACGTGCTCTGGAACGACTTTTTTCTTTTTCTTTAAAACAAAAAAAAGAAAGAAAAAAAAAACATTTACTACTACCAGTAGTACACCTCAACATTGGGTCCAGAACGTCCCAACTGCATGAGTCACTGGAGTCATGCCGAGGTCGCTAAGGTGCTGTAAAATACAACGTCAATTGAGAGAGACACAGGCGCAGCGCGCCGAGGGAGAAACGAGGCATTTATCTTCTGACCCTCCTTTTTACTCGTAATCTGTATCCCGGAACCGCGTCGCATCCATGTTAATTAAATCAACACTTACACTTGCTTGCTTCGTATGATGAAGATTTCTGACTGGCAACCCAGTCAGCAGCAGATTGGGGCAGATGTAGTAATGAAAAACACTGCAAGGTGTGACGTTTGAGACACTCCAATTGGTTAGAAAGCGACAAAGAAGACGTCGGAAAAATACCGGAAAAATCGAGTCTTTTTCTTTCTGCGTATTGGGCCCTTCTGCCTCCTTTGCCGCCCTTTCCACGCTCTTTCCACACCCTCACACTCCCTGAGCACTATGATCTCATTGCGCAATAAGATATACATGCACGTGCATTTGGTGAGCACGCAGAACCTTGTTGGGGGAAGATGCCCTAACCCTAAGGGCGTTCCATACGGTTCGACAGAGTAACCTTGCTGTCGATTATAACGCATATATAGCCCCCCCCTTCGGACCCTCCTTCTGATTTCTGTTTCTGTATCAACATTACACACAAACACACAA TGGTG

The foregoing detailed description has been given for clearness ofunderstanding only and no unnecessary limitations should be understoodthere from as modifications will be obvious to those skilled in the art.

While the disclosure has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the disclosure following, in general, theprinciples of the disclosure and including such departures from thepresent disclosure as come within known or customary practice within theart to which the disclosure pertains and as may be applied to theessential features hereinbefore set forth and as follows in the scope ofthe appended claims.

The disclosures, including the claims, figures and/or drawings, of eachand every patent, patent application, and publication cited herein arehereby incorporated herein by reference in their entireties.

What is claimed is:
 1. A method of producing a mono- or poly-unsaturatedC₆-C₂₄ fatty acyl-CoA, comprising: a) providing a recombinant Yarrowialipolytica comprising a heterologous nucleic acid molecule encoding andexpressing a Helicoverpa fatty acyl desaturase comprising atransmembrane desaturase sequence motif of HX₃₋₄HX₇₋₄₁(3 non-His)HX2-3(1non-His)HHX₆₁₋₁₈₉(40 non-His)HX₂₋₃(1 non-His)HH; and b) cultivating therecombinant Yarrowia lipolytica of (a) in a culture medium containing acarbon source feedstock and a saturated C₆-C₂₄ fatty acyl-CoA; whereinthe saturated C₆-C₂₄ fatty acyl-CoA is converted to a mono- orpoly-unsaturated C₆-C₂₄ fatty acyl-CoA by catalytic activity of thedesaturase.
 2. The method of claim 1, wherein the Helicoverpa fatty acyldesaturase is a Z11 desaturase.
 3. The method of claim 1, wherein themono- or poly-unsaturated C6-C24 fatty acyl-CoA is selected from thegroup consisting of Z11-13:Acyl-CoA, E11-13:Acyl-CoA,(Z,Z)-7,11-13:Acyl-CoA, Z11-14:Acyl-CoA, E11-14:Acyl-CoA,(E,E)-9,11-14:Acyl-CoA, (E,Z)-9,11-14:Acyl-CoA, (Z,E)-9,11-14:Acyl-CoA,(Z,Z)-9,11-14:Acyl-CoA, (E,Z)-9,11-15:Acyl-CoA, (Z,Z)-9,11-15:Acyl-CoA,Z11-16:Acyl-CoA, E11-16:Acyl-CoA, (E,Z)-6,11-16:Acyl-CoA,(E,Z)-7,11-16:Acyl-CoA, (E,Z)-8,11-16:Acyl-CoA, (E,E)-9,11-16:Acyl-CoA,(E,Z)-9,11-16:Acyl-CoA, (Z,E)-9,11-16:Acyl-CoA, (Z,Z)-9,11-16:Acyl-CoA,(E,E)-11,13-16:Acyl-CoA, (E,Z)-11,13-16:Acyl-CoA,(Z,E)-11,13-16:Acyl-CoA, (Z,Z)-11,13-16:Acyl-CoA,(Z,E)-11,14-16:Acyl-CoA, (E,E,Z)-4,6,11-16:Acyl-CoA,(Z,Z,E)-7,11,13-16:Acyl-CoA, (E,E,Z,Z)-4,6,11,13-16:Acyl-CoA,Z11-17:Acyl-CoA, (Z,Z)-8,11-17:Acyl-CoA, Z11-18:Acyl-CoA,E11-18:Acyl-CoA, (Z,Z)-11,13-18:AcylCoA, (E,E)-11, 14-18:Acyl-CoA, andcombinations thereof.
 4. The method of claim 1, wherein the Helicoverpafatty acyl desaturase is a Z9 desaturase.
 5. The method of claim 1,wherein the mono- or poly-unsaturated C6-C24 fatty acyl-CoA is selectedfrom the group consisting of Z9-11:Acyl-CoA, Z9-12:Acyl-CoA,E9-12:Acyl-CoA, (E,E)-7,9-12:Acyl-CoA, (E,Z)-7,9-12:Acyl-CoA,(Z,E)-7,9-12:Acyl-CoA, (Z,Z)-7,9-12:Acyl-CoA, Z9-13:Acyl-CoA,E9-13:Acyl-CoA, (E,Z)-5,9-13:Acyl-CoA, (Z,E)-5,9-13:Acyl-CoA,(Z,Z)-5,9-13:Acyl-CoA, Z9-14:Acyl-CoA, E9-14:Acyl-CoA,(E,Z)-4,9-14:Acyl-CoA, (E,E)-9,11-14:Acyl-CoA, (E,Z)-9,11-14:Acyl-CoA,(Z,E)-9,11-14:Acyl-CoA, (Z,Z)-9,11-14:Acyl-CoA, (E,E)-9,12-14:Acyl-CoA,(Z,E)-9,12-14:AcylCoA, (Z,Z)-9,12-14:Acyl-CoA, Z9-15:Acyl-CoA,E9-15:Acyl-CoA, (Z,Z)-6,9-15:AcylCoA, Z9-16:Acyl-CoA, E9-16:Acyl-CoA,(E,E)-9,11-16:Acyl-CoA, (E,Z)-9,11-16:AcylCoA, (Z,E)-9,11-16:Acyl-CoA,(Z,Z)-9,11-16:Acyl-CoA, Z9-17:Acyl-CoA, E9-18:AcylCoA, Z9-18:Acyl-CoA,(E,E)-5,9-18:Acyl-CoA, (E,E)-9,12-18:Acyl-CoA, (Z,Z)-9,12-18:Acyl-CoA,(Z,Z,Z)-3,6,9-18:Acyl-CoA, (E,E,E)-9,12,15-18:Acyl-CoA,(Z,Z,Z)-9,12,15-18:Acyl-CoA, and combinations thereof.
 6. The method ofclaim 1, wherein the recombinant Yarrowia lipolytica comprises a secondheterologous nucleic acid molecule encoding and expressing a fattyalcohol forming fatty-acyl reductase that converts the a mono- orpoly-unsaturated C6-C24 fatty acyl-CoA into a corresponding mono- orpoly-unsaturated C6-C24 fatty alcohol.
 7. The method of claim 6, furthercomprising metathesizing the mono- or poly-unsaturated C6-C24 fattyalcohol with an unsaturated C3-C10 hydrocarbon, thereby forming anelongated mono- or polyunsaturated C6-C24 fatty alcohol, or a truncatedmono- or polyunsaturated C6-C24 fatty alcohol.
 8. The method of claim 6,wherein the mono- or poly-unsaturated C6-C24 fatty alcohol is selectedfrom the group consisting of (Z)-3-hexen-1-ol, (Z)-3-nonen-1-ol,(E)-2-decen-1-ol, (Z)-2-decen-1-ol, (E)-3-decen-1-ol, (Z)-3-decen-1-ol,(Z)-4-decen-1-ol, (E)-5-decen-1-ol, (Z)-5-decen-1-ol, (E)-8-decen-1-ol,(Z,Z)-4,7-decadien-1-ol, (Z)-3-dodecen-1-ol, (E)-5-dodecen-1-ol,(Z)-5-dodecen-1-ol, (E)-6-dodecen-1-ol, (E)-7-dodecen-1-ol,(Z)-7-dodecen-1-ol, (E)-8-dodecen-1-ol, (Z)-8-dodecen-1-ol,(E)-9-dodecen-1-ol, (Z)-9-dodecen-1-ol, (E)-10-dodecen-1-ol,(Z)-10-dodecen-1-ol, (Z,Z)-3,6-dodecadien-1-ol,(E,E)-5,7-dodecadien-1-ol, (E,Z)-5,7-dodecadien-1-ol,(Z,E)-5,7-dodecadien-1-ol, (E,Z)-7,9-dodecadien-1-ol,(Z,E)-7,9-dodecadien-1-ol, (Z,Z)-7,9-dodecadien-1-ol,(E,E)-8,10-dodecadien-1-ol, (E,Z)-8,10-dodecadien-1-ol,(Z,E)-8,10-dodecadien-1-ol, (Z,Z)-8,10-dodecadien-1-ol,(Z,E,E)-3,6,8-dodecatrien-1-ol, (Z,Z,E)-3,6,8-dodecatrien-1-ol,(Z,Z)-4,7-tridecadien-1-ol, (E)-3-tetradecen-1-ol,(Z)-3-tetradecen-1-ol, (E)-5-tetradecen-1-ol, (Z)-5-tetradecen-1-ol,(E)-7-tetradecen-1-ol, (Z)-7-tetradecen-1-ol, (Z)-8-tetradecen-1-ol,(E)-9-tetradecen-1-ol, (Z)-9-tetradecen-1-ol, (E)-11-tetradecen-1-ol,(Z)-11-tetradecen-1-ol, (Z,Z)-5,8-tetradecadien-1-ol,(E,E)-8,10-tetradecadien-1-ol, (Z,E)-8,10-tetradecadien-1-ol,(Z,E)-9,11-tetradecadien-1-ol, (Z,Z)-9,11-tetradecadien-1-ol,(Z,E)-9,12-tetradecadien-1-ol, (Z,Z)-9,12-tetradecadien-1-ol,(E,E)-10,12-tetradecadien-1-ol, (Z,Z)-10,12-tetradecadien-1-ol,(E)-8-pentadecen-1-ol, (Z)-8-pentadecen-1-ol,(Z,Z)-6,9-pentadecadien-1-ol, (E,Z)-8,10-pentadecadien-1-ol,(E)-5-hexadecen-1-ol, (Z)-5-hexadecen-1-ol, (E)-7-hexadecen-1-ol,(Z)-7-hexadecen-1-ol, (E)-9-hexadecen-1-ol, (Z)-9-hexadecen-1-ol,(E)-10-hexadecen-1-ol, (E)-11-hexadecen-1-ol, (Z)-11-hexadecen-1-ol,(E,E)-1,3-hexadecadien-1-ol, (E,Z)-4,6-hexadecadien-1-ol,(Z,Z)-7,10-hexadecadien-1-ol, (Z,E)-7,11-hexadecadien-1-ol,(Z,Z)-7,11-hexadecadien-1-ol, (E,E)-10,12-hexadecadien-1-ol,(E,Z)-10,12-hexadecadien-1-ol, (E,E)-11,13-hexadecadien-1-ol,(E,Z)-11,13-hexadecadien-1-ol, (Z,E)-11,13-hexadecadien-1-ol,(Z,Z)-11,13-hexadecadien-1-ol, (E,E,Z)-4,6,10-hexadecatrien-1-ol,(E,Z,Z)-4,6,10-hexadecatrien-1-ol, (E)-8-heptadecen-1-ol,(Z)-8-heptadecen-1-ol, (Z)-11-heptadecen-1-ol,(Z,Z)-8,10-heptadecadien-1-ol, (E)-9-octadecen-1-ol,(Z)-9-octadecen-1-ol, (E)-11-octadecen-1-ol, (Z)-11-octadecen-1-ol,(Z)-13-octadecen-1-ol, (E,Z)-2,13-octadecadien-1-ol,(Z,Z)-2,13-octadecadien-1-ol, (E,E)-5,9-octadecadien-1-ol,(E,E)-9,12-octadecadien-1-ol, (E,E,E)-9,12,15-octadecatrien-1-ol, andcombinations thereof.
 9. The method of claim 6, wherein the recombinantmicroorganism further comprises at least one endogenous or exogenousnucleic acid molecule encoding an acetyl transferase that converts theC6-C24 fatty alcohol into a corresponding C5-C24 fatty acetate.
 10. Themethod of claim 9, further comprising metathesizing the mono- orpoly-unsaturated C6-C24 fatty acetate with an unsaturated C3-C10hydrocarbon, thereby forming an elongated mono- or polyunsaturatedC6-C24 fatty acetate, or a truncated mono- or polyunsaturated C6-C24fatty acetate.
 11. The method of claim 9, wherein the mono- orpolyunsaturated C6-C24 fatty acetate is an insect pheromone.
 12. Themethod of claim 9, wherein the mono- or polyunsaturated C6-C24 fattyacetate is selected from the group consisting of (E)-2-decenyl acetate,(Z)-2-decenyl acetate, (Z)-3-decenyl acetate, (E)-4-decenyl acetate,(Z)-4-decenyl acetate, (E)-5-decenyl acetate, (Z)-5-decenyl acetate,(E)-7-decenyl acetate, (Z)-7-decenyl acetate, (E,E)-3,5-decadienylacetate, (Z,E)-3,5-decadienyl acetate, (Z,Z)-4,7-decadienyl acetate,(E)-2-undecenyl acetate, (Z)-5-undecenyl acetate, (Z)-7-undecenylacetate, (Z)-8-undecenyl acetate, (Z)-9-undecenyl acetate,(E)-3-dodecenyl acetate, (Z)-3-dodecenyl acetate, (E)-4-dodecenylacetate, (E)-5-dodecenyl acetate, (Z)-5-dodecenyl acetate,(Z)-6-dodecenyl acetate, (E)-7-dodecenyl acetate, (Z)-7-dodecenylacetate, (E)-8-dodecenyl acetate, (Z)-8-dodecenyl acetate,(E)-9-dodecenyl acetate, (Z)-9-dodecenyl acetate, (E)-10-dodecenylacetate, (Z)-10-dodecenyl acetate, (E,Z)-3,5-dodecadienyl acetate,(Z,E)-3,5-dodecadienyl acetate, (E,E)-4,10-dodecadienyl acetate,(E,E)-5,7-dodecadienyl acetate, (E,Z)-5,7-dodecadienyl acetate,(Z,E)-5,7-dodecadienyl acetate, (Z,Z)-5,7-dodecadienyl acetate,(E,E)-7,9-dodecadienyl acetate, (E,Z)-7,9-dodecadienyl acetate,(Z,E)-7,9-dodecadienyl acetate, (Z,Z)-7,9-dodecadienyl acetate,(E,E)-8,10-dodecadienyl acetate, (E,Z)-8,10-dodecadienyl acetate,(Z,E)-8,10-dodecadienyl acetate, (Z,Z)-8,10-dodecadienyl acetate,(E)-2-tridecenyl acetate, (Z)-2-tridecenyl acetate, (E)-3-tridecenylacetate, (E)-4-tridecenyl acetate, (Z)-4-tridecenyl acetate,(E)-6-tridecenyl acetate, (Z)-7-tridecenyl acetate, (E)-8-tridecenylacetate, (Z)-8-tridecenyl acetate, (E)-9-tridecenyl acetate,(Z)-9-tridecenyl acetate, (Z)-10-tridecenyl acetate, (E)-11-tridecenylacetate, (Z)-11-tridecenyl acetate, (E,Z)-4,7-tridecadienyl acetate,(Z,Z)-4,7-tridecadienyl acetate, (E,Z)-5,9-tridecadienyl acetate,(Z,E)-5,9-tridecadienyl acetate, (Z,Z)-5,9-tridecadienyl acetate,(Z,Z)-7,11-tridecadienyl acetate, (E,Z,Z)-4,7,10-tridecatrienyl acetate,(E)-3-tetradecenyl acetate, (Z)-3-tetradecenyl acetate,(E)-5-tetradecenyl acetate, (Z)-5-tetradecenyl acetate,(E)-6-tetradecenyl acetate, (Z)-6-tetradecenyl acetate,(E)-7-tetradecenyl acetate, (Z)-7-tetradecenyl acetate,(E)-8-tetradecenyl acetate, (Z)-8-tetradecenyl acetate,(E)-9-tetradecenyl acetate, (Z)-9-tetradecenyl acetate,(E)-10-tetradecenyl acetate, (Z)-10-tetradecenyl acetate,(E)-11-tetradecenyl acetate, (Z)-11-tetradecenyl acetate,(E)-12-tetradecenyl acetate, (Z)-12-tetradecenyl acetate,(E,E)-3,5-tetradecadienyl acetate, (E,Z)-3,5-tetradecadienyl acetate,(Z,E)-3,5-tetradecadienyl acetate, (E,Z)-3,7-tetradecadienyl acetate,(E,Z)-3,8-tetradecadienyl acetate, (E,Z)-4,9-tetradecadienyl acetate,(E,Z)-4,10-tetradecadienyl acetate, (Z,Z)-5,8-tetradecadienyl acetate,(E,E)-8,10-tetradecadienyl acetate, (E,Z)-8,10-tetradecadienyl acetate,(Z,E)-8,10-tetradecadienyl acetate, (E,E)-9,11-tetradecadienyl acetate,(E,Z)-9,11-tetradecadienyl acetate, (Z,E)-9,11-tetradecadienyl acetate,(Z,Z)-9,11-tetradecadienyl acetate, (E,E)-9,12-tetradecadienyl acetate,(Z,E)-9,12-tetradecadienyl acetate, (Z,Z)-9,12-tetradecadienyl acetate,(E,E)-10,12-tetradecadienyl acetate, (E,Z)-10,12-tetradecadienylacetate, (Z,E)-10,12-tetradecadienyl acetate,(Z,Z)-10,12-tetradecadienyl acetate, (E,Z,Z)-3,8,11-tetradecatrienylacetate, (E)-8-pentadecenyl acetate, (Z)-8-pentadecenyl acetate,(Z)-9-pentadecenyl acetate, (E)-9-pentadecenyl acetate,(Z)-10-pentadecenyl acetate, (E)-12-pentadecenyl acetate,(Z)-12-pentadecenyl acetate, (Z,Z)-6,9-pentadecadienyl acetate,(E,E)-8,10-pentadecadienyl acetate, (E,Z)-8,10-pentadecadienyl acetate,(Z,E)-8,10-pentadecadienyl acetate, (Z,Z)-8,10-pentadecadienyl acetate,(Z)-3-hexadecenyl acetate, (E)-5-hexadecenyl acetate, (Z)-5-hexadecenylacetate, (E)-6-hexadecenyl acetate, (E)-7-hexadecenyl acetate,(Z)-7-hexadecenyl acetate, (E)-8-hexadecenyl acetate, (E)-9-hexadecenylacetate, (Z)-9-hexadecenyl acetate, (Z)-10-hexadecenyl acetate,(E)-11-hexadecenyl acetate, (Z)-11-hexadecenyl acetate,(Z)-12-hexadecenyl acetate, (Z)-14-hexadecenyl acetate,(E,Z)-4,6-hexadecadienyl acetate, (E,Z)-6,11-hexadecadienyl acetate,(Z,Z)-7,10-hexadecadienyl acetate, (Z,E)-7,11-hexadecadienyl acetate,(Z,Z)-7,11-hexadecadienyl acetate, (Z,Z)-8,10-hexadecadienyl acetate,(E,Z)-9,11-hexadecadienyl acetate, (E,E)-10,12-hexadecadienyl acetate,(E,Z)-10,12-hexadecadienyl acetate, (Z,E)-10,12-hexadecadienyl acetate,(E,E)-11,13-hexadecadienyl acetate, (E,Z)-11,13-hexadecadienyl acetate,(Z,E)-11,13-hexadecadienyl acetate, (Z,Z)-11,13-hexadecadienyl acetate,(Z,E)-11,14-hexadecadienyl acetate, (E,E,Z)-4,6,10-hexadecatrienylacetate, (E,Z,Z)-4,6,10-hexadecatrienyl acetate,(E,E,Z)-4,6,11-hexadecatrienyl acetate, (E,E,E)-10,12,14-hexadecatrienylacetate, (E,E,Z)-10,12,14-hexadecatrienyl acetate, (E)-8-heptadecenylacetate, (E)-10-heptadecenyl acetate, (Z)-11-heptadecenyl acetate,(E,E)-4,8-heptadecadienyl acetate, (Z,Z)-8,11-heptadecadienyl acetate,(E)-2-octadecenyl acetate, (Z)-2-octadecenyl acetate, (E)-9-octadecenylacetate, (Z)-9-octadecenyl acetate, (Z)-11-octadecenyl acetate,(E)-13-octadecenyl acetate, (Z)-13-octadecenyl acetate,(E,Z)-2,13-octadecadienyl acetate, (Z,E)-2,13-octadecadienyl acetate,(Z,Z)-2,13-octadecadienyl acetate, (E,E)-3,13-octadecadienyl acetate,(E,Z)-3,13-octadecadienyl acetate, (Z,E)-3,13-octadecadienyl acetate,(Z,Z)-3,13-octadecadienyl acetate, (E,E)-5,9-octadecadienyl acetate,(Z,Z)-9,12-octadecadienyl acetate, (Z,Z,Z)-3,6,9-octadecatrienylacetate, (Z,Z,Z)-9,12,15-octadecatrienyl acetate, and combinationsthereof.
 13. The method of claim 1, wherein the recombinant Yarrowialipolytica comprises a second heterologous nucleic acid moleculeencoding and expressing a fatty aldehyde forming fatty-acyl reductasethat converts the a mono- or poly-unsaturated C6-C24 fatty acyl-CoA to acorresponding mono- or poly-unsaturated C6-C24 fatty aldehyde.
 14. Themethod of claim 13, further comprising metathesizing the mono- orpoly-unsaturated C6-C24 fatty aldehyde with an unsaturated C3-C10hydrocarbon, thereby forming an elongated mono- or polyunsaturatedC6-C24 fatty aldehyde, or a truncated mono- or polyunsaturated C6-C24fatty aldehyde.
 15. The method of claim 13, wherein the mono- orpolyunsaturated C6-C24 fatty aldehyde is an insect pheromone.
 16. Themethod of claim 13, wherein the mono- or poly-unsaturated C6-C24 fattyaldehyde is selected from the group consisting of (E)-2-decenal,(Z)-2-decenal, (Z)-4-decenal, (Z)-5-decenal, (E,E)-2,4-decadienal,(E,Z)-2,4-decadienal, (Z,Z)-2,4-decadienal, (E)-2-undecenal,(E)-2-dodecenal, (Z)-5-dodecenal, (E)-6-dodecenal, (E)-7-dodecenal,(Z)-7-dodecenal, (E)-8-dodecenal, (E)-9-dodecenal, (Z)-9-dodecenal,(E)-10-dodecenal, (E,Z)-5,7-dodecadienal, (Z,E)-5,7-dodecadienal,(Z,Z)-5,7-dodecadienal, (E,Z)-7,9-dodecadienal, (E,E)-8,10-dodecadienal,(E,Z)-8,10-dodecadienal, (Z,E)-8,10-dodecadienal, (Z)-4-tridecenal,(E)-5-tetradecenal, (Z)-5-tetradecenal, (Z)-7-tetradecenal,(Z)-8-tetradecenal, (Z)-9-tetradecenal, (E)-11-tetradecenal,(Z)-11-tetradecenal, (E,E)-2,4-tetradecadienal,(E,Z)-4,9-tetradecadienal, (E,E)-5,8-tetradecadienal,(Z,Z)-5,8-tetradecadienal, (E,E)-8,10-tetradecadienal,(E,Z)-8,10-tetradecadienal, (Z,Z)-8,10-tetradecadienal,(Z,E)-9,11-tetradecadienal, (Z,Z)-9,11-tetradecadienal,(Z,E)-9,12-tetradecadienal, (E,E)-10,12-tetradecadienal,(Z)-10-pentadecenal, (Z,Z)-6,9-pentadecadienal,(E,Z)-9,11-pentadecadienal, (Z,Z)-9,11-pentadecadienal,(E)-7-hexadecenal, (Z)-7-hexadecenal, (E)-9-hexadecenal,(Z)-9-hexadecenal, (E)-10-hexadecenal, (Z)-10-hexadecenal,(E)-11-hexadecenal, (Z)-11-hexadecenal, (Z)-12-hexadecenal,(E)-14-hexadecenal, (E,Z)-4,6-hexadecadienal, (E,Z)-6,11-hexadecadienal,(Z,E)-7,11-hexadecadienal, (Z,Z)-7,11-hexadecadienal,(E,Z)-8,11-hexadecadienal, (E,E)-9,11-hexadecadienal,(E,Z)-9,11-hexadecadienal, (Z,E)-9,11-hexadecadienal,(Z,Z)-9,11-hexadecadienal, (E,E)-10,12-hexadecadienal,(E,Z)-10,12-hexadecadienal, (Z,E)-10,12-hexadecadienal,(Z,Z)-10,12-hexadecadienal, (E,E)-11,13-hexadecadienal,(E,Z)-11,13-hexadecadienal, (Z,E)-11,13-hexadecadienal,(Z,Z)-11,13-hexadecadienal, (E,E)-10,14-hexadecadienal,(E,E,Z)-4,6,11-hexadecatrienal, (Z,Z,E)-7,11,13-hexadecatrienal,(E,E,E)-10,12,14-hexadecatrienal, (E,E,Z)-10,12,14-hexadecatrienal,(E,E,Z,Z)-4,6,11,13-hexadecatetraenal, (E)-2-heptadecenal,(Z)-2-heptadecenal, (Z)-9-heptadecenal, (E)-2-octadecenal,(Z)-2-octadecenal, (E)-9-octadecenal, (Z)-9-octadecenal,(E)-11-octadecenal, (Z)-11-octadecenal, (E)-13-octadecenal,(Z)-13-octadecenal, (E)-14-octadecenal, (E,Z)-2,13-octadecadienal,(E,Z)-3,13-octadecadienal, (Z,Z)-3,13-octadecadienal,(Z,Z)-9,12-octadecadienal, (Z,Z)-11,13-octadecadienal,(E,E)-11,14-octadecadienal, (Z,Z)-13,15-octadecadienal,(Z,Z,Z)-9,12,15-octadecatrienal, and combinations thereof.
 17. Themethod of claim 1, wherein the heterologous nucleic acid moleculecomprises a nucleotide sequence encoding the amino acid sequence encodedby SEQ ID NO:
 39. 18. The method of claim 1, comprising a disruption ofthe fao1 gene.
 19. The method of claim 1, comprising a disruption of thepox1, pox2, pox3, pox4, pox5, and/or pox6 genes.
 20. The method of claim1, comprising a disruption of the adh1, adh2, adh3, adh4, adh5, adh6,and/or adh7 genes.
 21. The method of claim 1, wherein the heterologousnucleic acid molecule comprises a nucleotide sequence encoding an aminoacid sequence having at least 80% identity to the amino acid sequenceencoded by SEQ ID NO:
 27. 22. The method of claim 1, wherein theheterologous nucleic acid molecule comprises the nucleotide sequence ofSEQ ID NO: 27.